Physio-chemical classification of membrane proteins-a review
Dinesh kumar Gupta
Shree Gupta Institute, 903, Bai-ka-Bagicha,
jabalpur(M.P.)- 482001, India
Abstract
On the basis of shape/structure, function performed and localization in fluid matrix, the membrane protein are classified into six classes. Different proteins of well characterized erythrocyte membranes are classified under proposed physio-chemial classification. As there exists phylogenicially and structurally similar proteins in wide diversity of biomembranes, it is needed to develop a general classification of membrane proteins.
Key words: Biomembranes, amphitropic proteins, accreted proteins, linker proteins, modulator proteins, cytosolic functional proteins.
Introduction
Traditionally, membrane proteins were classified into intrinsic and extrinsic proteins. As new proteins were identified, terms like lipid anchored protein, acylated and (iso) prenylated proteins were used. Intrinsic proteins were later on classified into type I,II,III & IV while acylated proteins are classified as type V. The transmembrane GPI anchored proteins (dually attached ) were classified as type VI proteins . In the present classification of membrane proteins, these are classified into six classes. The different classes of membrane proteins are cytosolic structural proteins, cytosolic functional proteins, linker proteins, amphitropic proteins, structural and functional transmembrane proteins. The criteria of classification is illustrated by quoting the example of erythrocyte membrane proteins, As there exists proteins similar to that of erythrocyte in different non-erythroid cells, this classification can be accepted as general classification of membrane proteins.
Dynamic state of membrane is due to the presence of diverse types of protein molecules. Proteins exists in different configuration by acquiring a wide variety of conformation. The globular conformation dominates the expanded conformation and is interdigitated between the hydrophobic domain of lipid bilayer by means of hydrophobic interaction or by means of covalent bonds1. On freeze fracture microscopy, some of these proteins exists as intra-membranous particles2. These globular intra-membranous particles are important functional complexes of dynamic biomembrane and are involved in the transport of osmolytes, solutes, metabolites and targeting of peptides, as well as proper orientation of receptor and adaptor molecules. Contrary to globur proteins, the expanded conformations possessing macromolecules form the immunogenic domain, membranoskeleton, cytoskeleton3 etc. and are important macromolecular complexes in discriminating between self and non-self, cellular shape and biomembrane macromolecular architecture in the three dimensional spatiotemporal frame - work.
Existing system of classification
On the basis of solubility of membrane proteins i.e. the type of solution required to disintegrate and extract proteins from their hydrophobic domain, the membrane proteins were traditionally classified as extrinsic (peripheral) and intrinsic(integral) proteins4. In 1981, Houslay had classified membrane proteins into six classes out of which class I - to class IV were integral protein and class V and class VI were peripheral proteins. Class I includes transmembrane globular integral proteins, class II transmembrane fibrous integral protein, class I I I globular protein in aqueous phase but anchored by an hydrophilic pedicle that does not span the bilayer while that of class IV includes globular integral proteins associated essentially with one or other half of the bilayer. In Houslay system of classification, class V were peripheral proteins, associated essentially through interaction with phospholipids while class VI were peripheral proteins capable of binding to specific integral transmembrane proteins. The above system of nomenclature does not gain prominence among membranologists as it had given more emphasis on the physico-chemical properties of the protein rather than to the range of functions that a particular protein performs in the functional membrane. Ramasarma6 used the term lipid anchored proteins to all those proteins which were covalently attached with the constituent of hydrophobic domain of membrane by forming covalent linkage with the hydrophobic chains of fatty acids. Gupta7 while reviewing membrane proteins have classified it into six classes on the basis of (a) the function performed by protein to maintain dynamic state of membrane and (b) the spatial localization of protein in the three dimensional hydrophobic domain. Singer8 has further classified single hydrophobic domain containing integral protein as type I and II while proteins possessing multiple hydrophobic domain as type III and IV . The acylated and GPI anchored protein were termed as type V9 and transmembrane GPI anchored proteins as type VI10 . These membrane anchored transmembrane proteins are the cell surface protein 'ponticulin'11 and cysteine rich hydrophobic Sm 23 protein, a surface protein of human parasite Schistosoma mansoni 12 . Extensive analysis of erythroid and noneythroid membrance proteins of normal, pathological and different physiological state as well as in vivo and in vitro manipulation technuques 13 have generated diverse range of data and is expected to generate more information on some unexplored physio-chemical nature of these proteins and macromolecular complexes. Thus, the existing system of classification needs to be modified and elaborated.
Tenets and concept of physio-chemical classification
To achieve the above task, the basic tenet proposed for physio-chemical classification of membrane protein is that, the range of functional and structural niche occupied by protein is not only dependent on the physico-chemical sturcture of protein but also on the localization of proteins in the spatio-temporal frame work of hydrophobic domain of membrane existing in the dynamic state. This is well illustrated by the fact that the isoprenylation(farnesylation, geranyl-geranylation) of g protein 14 (modulator component of modulator subsysem15) and other ras related protein is a critical step for sub-cellular localization and acquisition of biological activities of the cell growth and regulation. The other useful concept is the existence of three heterogenous domains in the functional membrane which were termed as extracellular hydrophilic environment, lipophilic-hydrophobic environment and cytosolic-hydrophilic environment 15. The above mentioned physio-chemical tenets and the concept of existence of heterogeneous domain in a functional biomembrane are the basis of the physio-chemical classification of the membrane proteins.
Physio-chemical classes of membrane proteins
Erythrocyte membrane proteins are classified into six classes on the basis of range of functions performed by the proteins to maintain dynamic state of erythrocyte, as well as spatial localization of the proteins as observed by different techniques, viz., microscopic 16, electrophoretic 17 and immunogenic labeling analysis. The six classes of erythrocyte membrane proteins are i) cytosolic structural proteins ii) cytosolic functional proteins iii) linker proteins, iv) amphitropic proteins, v) structural transmembrane proteins and vi) functional transmembrane proteins.
Table-1 summarizes the proposed classification of well characterized erythrocyte membrane proteins. The criteria used to classify membrane pr4oteins into six classes are elaborated by illustrating the example of erythrocyte membrane proteins or their macromolecular content.
No. Classes Erythroid Proteins Physio-chemical characteristics
1 Cytosolic structural proteins Spectrin α Soluble in neutral aqueous buffer as they are totally free from phospholipids and can be solubilized by altering ionic strength of the medium or by adding chelating agents.
Spectrin β
Actin gelatinous
Actin filamentous
2 Cytosolic functional proteins Glyceraldehyde-3-phosphate,dehydrogenase, catalase, membrane bound hexokinase & other enzymes Gets accreted with the hydrophobic domain in presence of ionic species or generation of metabolite and redox potential during different physiological and pathophysiological state.
3 Linker proteins Ankyrin, Band 4.1 Soluble in netural salt solution and generally exist as an acylated form can be solubilized from the membrane by altering ionic strength and pH of the medium.
4 Amphitropic proteins Acetylcholinesterases The functional proteins whose characteristic anre altered i.e. affinity for substrate or Kd when solubilized from the membrane by the enzymatic action, high salt or ionic detergent containing buffer.
Acid and alkaline phosphatases
Peptides of G proteins
5 Structural transmembrane proteins Glycophorin C The single hydrophobic domain containing integral peptides generally remain associated with phospholipids when extracted from membrane.
6 Functional transmembrane proteins Anion transporters, Cation dependent ATPases, Receptors of different ligands These are generally multimeric hydrophobic domain containing intergrl proteins when extracted exist as protein-phospholipid particulate complexes and are dependent on a particular hydrophobic requirement to execute their function.
Cytosolic structural proteins:
These proteins are extrinsic membrane proteins18 having the following characteristics:
a) They require only mild treatment such as the alteration in the ionic strength of the medium or the addition of a chelating agent to dissociate them moleculary from the membranes:
b) They dissociate totally from lipids, and
c) In the dissociated state they are relatively soluble in neutral aqueous buffers.
Spectrin and actin come under this group. Spectrin is the most abundant cytosolic structural protein in erythroid and non-erythroid cells. Human erythrocytes membrane spectrin is composed of 240 kDa alpha (α or band 1) and 225 kDa beta(β or band 2) peptide subunits. The molecular diversity and evolution of erythroid and non-erythroid spectrin had already been elaborated 19.20 . These a and b subunits can form homodiamer i.e. (α)2 and (β)2 in pathological states, heterodimers i.e. (αβ ) and tetramer(αβ) 2 in normal physiological state, while other oligomers are also observed in solutions. In heterodimer, the amino terminal of β subunit aligned with carboxy terminal of α subunit parallel to one another. These dimers of spectrin self associate in the region containing 80 kDa segment that begins at the amino terminal of the α subunit . Besides self associating segment, human and other animal erythrocyte membrane spectrin contains specific binding sites for ankyrin, actin, band 4.1, calmodulin 21 and the metabolite 2.3. diphosphoglycerate 22-24 . The subunits of spectrin forming the head group have ankyrin binding segment, while tail of α/ β spectrin dimer, actin and band 4.1 binds and forms the meshwork of protein. Actin exists in two form i.e. G actin and F actin. The polymerized and filamentous form of F actin is the constituents of membranoskeleton junctional complexes. The major protein constituents of membranoskeletal complex are tail region of α/ β spectrin dimer, band 4.1 and actin while myosin, tropomyosin and band 4.9 are the minor component stablizing the F actin -F actin, actin-spectrin interaction.
Cytosolic functional proteins.
Cytosolic functional proteins partition themselves between the cytosol membrane interface (cytosolic hydrophilic/hydrophobic environement ) in order to maintain an optimum state. These are membrane accreted proteins having following characteristics:
a) These proteins interact with the membrane at the cytosol - membrane interface/ cytosolic gydrophobic-hydrophilic environment.
b) the interaction is dependent on the ionic, metabolic stress and stain status of the cells.
c) In the dissociated state, the kihnetic constant like Km, Ki and Vmax of functional enzymes or affinity for ligands in case of enzymes or modulators is different than that in membrane bound state.
Accreted proteins are mainly the enzyme of glycolytic and redox pathway. They maintain the steady state of carbon flux and redox states for the generation of ATP by substrate level phosphorylation by altering the activity of key enzymes as reported in mild insulin dependent diabetes mellitus rat erythrocytes 25,26 . To maintain an optimum content, some of the enzymes particularly glyceraldehyde-3- phosphate dehydrogenase (G-3-PD) accrete with the membrane as observed on SDS-PAGE electrophoretogram 27 which is designated as band 6 proteins 17, while catalase accrete during oxidant stress. The accretion of G-3-PD and other enzymes is dependent on the ionic stength of the cytosol. Number of other funtional peptide accrete with the cytosolic segment of band 3 proteins. Accretion of cylindrin-a cylindrical acidic protein of unknown function and calmodulin are other examples. Cylindrin has been reported both in membrane and cytosolic fraction 28.29 while the erythrocyte calmodulin interacts with membrane via spectrin and Ca2+- ATPase cytosolic peptide.
Linker Proteins
Linker proteins link the extrinsic proteins with the intrinsic transmembrane proteins. These are extrinsic acylated (palmitoylated, myristolated) proteins having the following characteristics:
a) These proteins require ionic treatment only, such as high ionic strength/or strongly alkaline pH media devoid of nay detergent to dissociate them molecularly from the membrane.
b) in the dissociated stae, traces of either acylated or triphosphoinositoide moiety is present.
c) These proteins are soluble in neutral salt solution.
Ankyrin and band 4.1 are the major acylated linker proteins in erythrocyte membranes.
Ankyrin
Ankyrin link the specific segment of β spectrin subunit with major transmembrane glycoprotein i.e., band 3 It is a multiple phosphorylated asymmetric protein of 210 kDa in human erythrocyte membrane and is first protein to be identified to have a high-affinity binding site for spectrin on the inner surface of bilayer. Ankyrin has two distinet co-operative binding site for the erythrocyte anion exchanger 30 out of wihich, one site is salt sensitive which suggests that the interaction is mediated by electrostatic interaction, while the other site was not salt sensitive, indicating that the interaction is likely to be mediated by hydrogen bonding or hydrophobic contacts. Ankyrin is an acylated protein mainly having the palmitic acid residues at the band 3 binding segment31.
In terms of binomial numerology, it is known as band 2.1 and is susceptible to ca 2+ activated proteases. In pathophysiological and experimental states13 having high intracellular Ca2+ contents, different bands are observed between band 2 and 3 and are termed as band 2.1- 2.6. These bands are collectively known as syndeins 32. Reduction in ankyrin disrupts the linkage with the anion exchanger which destabilize the membrane associated with serve hemolytic anemia 33. Besides stablizing the membrane, the human erythrocyte ankyrin contain a 62kDa spectrin binding domain which is antigenically and structurally similar to P57, a membrane serine proteinase34.35.
Band 4.1 proteins
Protein 4.1 is composed of two identical 80 and 78 KDa peptide, the former differs from the later in carboxy terminal amino acid residues. Band 1.1 protein differs from ankyrin in term that it does not bind to a specific segment of one of the spectrin subunits, but instead requires the presence of both subunits to generate a stable complex comprising spectrin, band 4.1, actin, tropomyosin and band 4.9, it links the membranoskeleton with transmembrane glycophorin A as well as glycophorin C36 . The binding of band 4.1 with glycophorins is mediated by tri-phosphoinositoides 37. The altered band 4.1a/4.1b content observed on SDS_PAGE by using discontinuous buffer system of laemmli is explored by gerontologists to explain senescence of erythrocytes 38.
Amphitropic proteins
These proteins are linked to the membrane via some modified phospholipids constituents of the bilayer. One of the widely studied bilayer constituents is glycatedphosphatidyl inositol (GPI) having different moiety of carbohydrate attached to it in different animals 39. Some of the cell surface membrane proteins like ponticulin 11 that forms the major high affinity link between the plasma membrane and actin cortical network and Sn 23 protein12-a cell surface protein found on the human gut parasite S. manosi, were GPI anchored transmembrane proteins. The amphitropic proteins have the following characteristics:
a) These proteins can be dissociated from the bilayer either by the activation of a specific enzymes or high salt containing buffer system or by using ionic detergent.
b) They can acquire different conformers which depend on the constituents of the solution used to dissociate them from the bilayer.
c) The kinetic constant i.e. Vmax,km,Ki and affinity of enzymes differ when they exist as soluble form (i.e. dissociated state) from that of particulate form.
Erythrocyte membrane acetylcholinesterase (AChE), acid and alkaline phosphatases are the classical example of extracellular exofacial amphitropic proteins while different peptide constituent of functional G protein are example of cytosolic endofacial amphitropic proteins. Earlier AChE has been classified as an extrinsic as well as intrinsic proteins 40 which depends on the buffer system used to soublilize it. Different species of erythrocyte have different derivative of glycosylphosphatidyl inositol (GPI) due to which the response of bilayer to phosphoinositol phospholipase C(PI-PLC) to release AChE varies from species to species 41. The in-depth analysis of the sequence of event associated with the release of AChE due to PI-PLC activation will help us to understand precisely the biological significance of AChE in non-nucleated erythrocytes while acid and alkaline phosphatases cintent in serum is extensively used to evaluate the effect of toxicanst on different biological organization. The ubiquious occurrence of these proteins in biological system and their cellular and molecular biology has been extensively reviewed 42. The relevance of these proteins in evolution has also been elaborated43.
Structural transmembrane proteins
The structural tranmembrane proteins are integral membrane proteins generally having a single, 22-25 amino acid residues containing hydrophobic domain. The characteristic feature of these proteins are:
a) They require drastic treatment with reagents such as detergens, bile acids, protein denaturanst or organic solvent to dissociate them from hydrophobic domain of biomembranes.
b) in many instances the functional proteins remain associated with lipids when isolated.
c) If completely dissociated from lipids, they are usually highly insoluble or aggregated in neutral aqueous buffers thus having an altered cooperatively with its ligands.
Different glycophorins are major structural transmembrane proteins in erythrocyte membrane. The glycoproteins constituting transmembrane proteins differ in the length and complexity of the oligosaccharide chains and the proportions of sialic acid residues attached to them . These are detected by PAS staining of the SDS-PAGE gels. The glycoproteins are characterized on the basis of amino acid carbohydrate and sialic acid residues into glycophorin A(GPA), glycophorin B(GPB) , glycophorin C (GPC) and glycophorin D(GPD)44. Glycophorin A is a also known as PASI, GPB as PASIII while GPC as PASII . These glycophorins contain a single membrane spanning segment of 22-23 uncharged amino acids just-long enough to cross the non-polar region of the bilayer having N terminal on the extracellular side Glycophorin A is the carrier of MN blood group while glycophorin B is the carrier of Ss blood group. Glycophorin C consists different variant of β sialoglycopeptides while glycophorin D contains α sialoglycopeptides only. On the contrary, the ABO, Rh and lewis blood group of human are determined by glycosphingolipids. Glycophorin C and D is responsible for leech phenotype lacking the common Gebrich blood group. In the elliptocytes of homozygous Gerbich blood group dieficient individual, the relative lack of linker protein 4.1 has also been observed 45. The analysis of these mutant erythrocyte membrane organization supported that glycophorin C possesses the binding site for protein 4.1 and tus is essential fro the maintenance of discocyte shape.
Functional transmembrane proteins
The functional transmembrane proteins are either multimeric complex or oligomeric integral proteins generally having 45-220 amino-acid residues containning 2-7 hydrophobic domain spanning segment each having 22-25 amino acid residues. The characteristic feature of these protein are;
a) They require drastic treatment with reagents such as detergents, bile acids, proteins denaturants or organic solvent to dissociate them from hydrophobic domain of biomembranes.
b) In many instances, the functional proteins exist in the form of protein phospholipid particulate complex.
c) If ciompletely dissociated from lipids, they are usually highly insoluble or aggregated in neutral aqueous buffers thus having an altered cooperativity with its ligands or are rendered functionless due to the loss of their capability to phosphorylate their specific amino-acid residues.
The cation dependent ATPases, anion and glucose transporters exists in the form of protein phospholipids particulate compleses and are generally made up of multimers of subunits occasionally capable of forming transmembrane channels. The cation dependent ATPases and transporters maintain the optimum contents of osmotically and physiologically necessary ionic species.
Anion and glucose transporter
The heterogeneous protein of about 90.0 kDa is involved in the exchange of chloride, bicarbonate and water across the red cell membranes. It is constituted by a group of heterogeneous polypeptides known as band 3 protein . Band 3 is glycosylated in extracellular surface while the cytosolic domain possesses the binding site of haemoglobin, glycolytic enzymes 46 and the linker protein i.e. ankyrin. The site of band 3 polypeptides involved in binding with ankyrin (band 2.1) maintains the structural integrity of erythrocytes by linking membranoskeleton with the hydrophobic domain . Band 3 protein is also the precursor of 62 kDa senescent autontigen 47 as well as band 4.5 the glucose tranporter 48. It is believed that the senescent autoantigen and glucose tranporter are produced by the proteolytic degradation of anion transporter proteins. The anion transporter proteins have multiple polypeptide chains across the lipid bilayer. These polypeptides have charged amino acids. The side chain of specific amino acid pair with specific amino acids to form aqueous channel across the lipid bilayer . As these aqueous channels are involved in the transfer of anion, the polypeptides constituting it are called anion channel proteins (ACP) . The ACP is main component of band 3 and is made up of two distinct segments: i) a membrane spanning segment, facilitating the exchange of anion across the membranes; ii) a cytoplasmic segment which is the locus of multiple interactions between the integral domain of the proteins, the cytoskeleton and cytosolic proteins.
The functional ACP is a phosphoprotein phosphorylated by caseinkinases ands band 3 tyrosine kinase. Vasseur49 reoorted that when band 3 is phosphorylated in the presence of Mg24 51% of phosphorylated amine acid is tyrosine,while phosphorylated serine and phosphotheorinine are 34% and 15% respectively. Phosphorylation of the cytoplasmic segment regulates the interaction of ACP with cytosolic structural and functional proteins.
The other band 3 proteins are glycophorin A having a single polypeptide of about 22-23 neutral amino-acids in membrane spanning segment while Ca 2+ ATPase is another protein. All the proteins of band 3 are glycosylated to varying degree and undrgo phosphorylation to execute their function. Cation dependent ATPases are elaborately discussed and described earlier7. Although, erythroid insulin receptors and many other receptors of hormones, growth factors, cytokinins etc. have been characterized. The receptors and many other receptors of hormones, growth factors, cytokinins etc. have been characterized. The receptors of other ligands present in extra-cellular hydrophilic environment of erythrocyte membrane along with the enzymatic mechanism of isoprenylation 50 and accretion of different G peptides 51 at cytosolic hyrophobic/hydrophilic interface need to be specifically analyzed and understood. After extensive structural and functional analysis and the subjective and intuitive understanding of the localization of receptors and various G peptide in varied dynamic state, and attempt should be made to classify the above mentioned proteins to any of the six classes proposed in physio-chemical classification of membrane proteins.
In different non-erythroid cell diverse type of proteins which are functionally similar to that of erythroid cell have been reported and extensively reviewed in case of erythroid spectrin and non-erythroid fordin 19.52.53 Thus, the criteria proposed above to group membrane proteins into six classes may be extensively used for classifying membrane proteins isolated from different cell and organs. Unanimous acceptance of above classification may provide impetus to develop biomembranology as a separate multidisciplinary discipline of basic and applied biological sciences.
Acknowledgement
Author express his gratitude to Prof. J,S. Rathore and Prof. M. Suhail for constant encouragement and to the Principal Trustee, Shri Gupta Institute, Jabalpur, for enduring financial support.
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Tuesday, May 4, 2010
Physio-chemical classification of membrane proteins-a review
Dinesh kumar Gupta
Shree Gupta Institute, 903, Bai-ka-Bagicha,
jabalpur(M.P.)- 482001, India
Abstract
On the basis of shape/structure, function performed and localization in fluid matrix, the membrane protein are classified into six classes. Different proteins of well characterized erythrocyte membranes are classified under proposed physio-chemial classification. As there exists phylogenicially and structurally similar proteins in wide diversity of biomembranes, it is needed to develop a general classification of membrane proteins.
Key words: Biomembranes, amphitropic proteins, accreted proteins, linker proteins, modulator proteins, cytosolic functional proteins.
Introduction
Traditionally, membrane proteins were classified into intrinsic and extrinsic proteins. As new proteins were identified, terms like lipid anchored protein, acylated and (iso) prenylated proteins were used. Intrinsic proteins were later on classified into type I,II,III & IV while acylated proteins are classified as type V. The transmembrane GPI anchored proteins (dually attached ) were classified as type VI proteins . In the present classification of membrane proteins, these are classified into six classes. The different classes of membrane proteins are cytosolic structural proteins, cytosolic functional proteins, linker proteins, amphitropic proteins, structural and functional transmembrane proteins. The criteria of classification is illustrated by quoting the example of erythrocyte membrane proteins, As there exists proteins similar to that of erythrocyte in different non-erythroid cells, this classification can be accepted as general classification of membrane proteins.
Dynamic state of membrane is due to the presence of diverse types of protein molecules. Proteins exists in different configuration by acquiring a wide variety of conformation. The globular conformation dominates the expanded conformation and is interdigitated between the hydrophobic domain of lipid bilayer by means of hydrophobic interaction or by means of covalent bonds1. On freeze fracture microscopy, some of these proteins exists as intra-membranous particles2. These globular intra-membranous particles are important functional complexes of dynamic biomembrane and are involved in the transport of osmolytes, solutes, metabolites and targeting of peptides, as well as proper orientation of receptor and adaptor molecules. Contrary to globur proteins, the expanded conformations possessing macromolecules form the immunogenic domain, membranoskeleton, cytoskeleton3 etc. and are important macromolecular complexes in discriminating between self and non-self, cellular shape and biomembrane macromolecular architecture in the three dimensional spatiotemporal frame - work.
Existing system of classification
On the basis of solubility of membrane proteins i.e. the type of solution required to disintegrate and extract proteins from their hydrophobic domain, the membrane proteins were traditionally classified as extrinsic (peripheral) and intrinsic(integral) proteins4. In 1981, Houslay had classified membrane proteins into six classes out of which class I - to class IV were integral protein and class V and class VI were peripheral proteins. Class I includes transmembrane globular integral proteins, class II transmembrane fibrous integral protein, class I I I globular protein in aqueous phase but anchored by an hydrophilic pedicle that does not span the bilayer while that of class IV includes globular integral proteins associated essentially with one or other half of the bilayer. In Houslay system of classification, class V were peripheral proteins, associated essentially through interaction with phospholipids while class VI were peripheral proteins capable of binding to specific integral transmembrane proteins. The above system of nomenclature does not gain prominence among membranologists as it had given more emphasis on the physico-chemical properties of the protein rather than to the range of functions that a particular protein performs in the functional membrane. Ramasarma6 used the term lipid anchored proteins to all those proteins which were covalently attached with the constituent of hydrophobic domain of membrane by forming covalent linkage with the hydrophobic chains of fatty acids. Gupta7 while reviewing membrane proteins have classified it into six classes on the basis of (a) the function performed by protein to maintain dynamic state of membrane and (b) the spatial localization of protein in the three dimensional hydrophobic domain. Singer8 has further classified single hydrophobic domain containing integral protein as type I and II while proteins possessing multiple hydrophobic domain as type III and IV . The acylated and GPI anchored protein were termed as type V9 and transmembrane GPI anchored proteins as type VI10 . These membrane anchored transmembrane proteins are the cell surface protein 'ponticulin'11 and cysteine rich hydrophobic Sm 23 protein, a surface protein of human parasite Schistosoma mansoni 12 . Extensive analysis of erythroid and noneythroid membrance proteins of normal, pathological and different physiological state as well as in vivo and in vitro manipulation technuques 13 have generated diverse range of data and is expected to generate more information on some unexplored physio-chemical nature of these proteins and macromolecular complexes. Thus, the existing system of classification needs to be modified and elaborated.
Tenets and concept of physio-chemical classification
To achieve the above task, the basic tenet proposed for physio-chemical classification of membrane protein is that, the range of functional and structural niche occupied by protein is not only dependent on the physico-chemical sturcture of protein but also on the localization of proteins in the spatio-temporal frame work of hydrophobic domain of membrane existing in the dynamic state. This is well illustrated by the fact that the isoprenylation(farnesylation, geranyl-geranylation) of g protein 14 (modulator component of modulator subsysem15) and other ras related protein is a critical step for sub-cellular localization and acquisition of biological activities of the cell growth and regulation. The other useful concept is the existence of three heterogenous domains in the functional membrane which were termed as extracellular hydrophilic environment, lipophilic-hydrophobic environment and cytosolic-hydrophilic environment 15. The above mentioned physio-chemical tenets and the concept of existence of heterogeneous domain in a functional biomembrane are the basis of the physio-chemical classification of the membrane proteins.
Physio-chemical classes of membrane proteins
Erythrocyte membrane proteins are classified into six classes on the basis of range of functions performed by the proteins to maintain dynamic state of erythrocyte, as well as spatial localization of the proteins as observed by different techniques, viz., microscopic 16, electrophoretic 17 and immunogenic labeling analysis. The six classes of erythrocyte membrane proteins are i) cytosolic structural proteins ii) cytosolic functional proteins iii) linker proteins, iv) amphitropic proteins, v) structural transmembrane proteins and vi) functional transmembrane proteins.
Table-1 summarizes the proposed classification of well characterized erythrocyte membrane proteins. The criteria used to classify membrane pr4oteins into six classes are elaborated by illustrating the example of erythrocyte membrane proteins or their macromolecular content.
No. Classes Erythroid Proteins Physio-chemical characteristics
1 Cytosolic structural proteins Spectrin α Soluble in neutral aqueous buffer as they are totally free from phospholipids and can be solubilized by altering ionic strength of the medium or by adding chelating agents.
Spectrin β
Actin gelatinous
Actin filamentous
2 Cytosolic functional proteins Glyceraldehyde-3-phosphate,dehydrogenase, catalase, membrane bound hexokinase & other enzymes Gets accreted with the hydrophobic domain in presence of ionic species or generation of metabolite and redox potential during different physiological and pathophysiological state.
3 Linker proteins Ankyrin, Band 4.1 Soluble in netural salt solution and generally exist as an acylated form can be solubilized from the membrane by altering ionic strength and pH of the medium.
4 Amphitropic proteins Acetylcholinesterases The functional proteins whose characteristic anre altered i.e. affinity for substrate or Kd when solubilized from the membrane by the enzymatic action, high salt or ionic detergent containing buffer.
Acid and alkaline phosphatases
Peptides of G proteins
5 Structural transmembrane proteins Glycophorin C The single hydrophobic domain containing integral peptides generally remain associated with phospholipids when extracted from membrane.
6 Functional transmembrane proteins Anion transporters, Cation dependent ATPases, Receptors of different ligands These are generally multimeric hydrophobic domain containing intergrl proteins when extracted exist as protein-phospholipid particulate complexes and are dependent on a particular hydrophobic requirement to execute their function.
Cytosolic structural proteins:
These proteins are extrinsic membrane proteins18 having the following characteristics:
a) They require only mild treatment such as the alteration in the ionic strength of the medium or the addition of a chelating agent to dissociate them moleculary from the membranes:
b) They dissociate totally from lipids, and
c) In the dissociated state they are relatively soluble in neutral aqueous buffers.
Spectrin and actin come under this group. Spectrin is the most abundant cytosolic structural protein in erythroid and non-erythroid cells. Human erythrocytes membrane spectrin is composed of 240 kDa alpha (α or band 1) and 225 kDa beta(β or band 2) peptide subunits. The molecular diversity and evolution of erythroid and non-erythroid spectrin had already been elaborated 19.20 . These a and b subunits can form homodiamer i.e. (α)2 and (β)2 in pathological states, heterodimers i.e. (αβ ) and tetramer(αβ) 2 in normal physiological state, while other oligomers are also observed in solutions. In heterodimer, the amino terminal of β subunit aligned with carboxy terminal of α subunit parallel to one another. These dimers of spectrin self associate in the region containing 80 kDa segment that begins at the amino terminal of the α subunit . Besides self associating segment, human and other animal erythrocyte membrane spectrin contains specific binding sites for ankyrin, actin, band 4.1, calmodulin 21 and the metabolite 2.3. diphosphoglycerate 22-24 . The subunits of spectrin forming the head group have ankyrin binding segment, while tail of α/ β spectrin dimer, actin and band 4.1 binds and forms the meshwork of protein. Actin exists in two form i.e. G actin and F actin. The polymerized and filamentous form of F actin is the constituents of membranoskeleton junctional complexes. The major protein constituents of membranoskeletal complex are tail region of α/ β spectrin dimer, band 4.1 and actin while myosin, tropomyosin and band 4.9 are the minor component stablizing the F actin -F actin, actin-spectrin interaction.
Cytosolic functional proteins.
Cytosolic functional proteins partition themselves between the cytosol membrane interface (cytosolic hydrophilic/hydrophobic environement ) in order to maintain an optimum state. These are membrane accreted proteins having following characteristics:
a) These proteins interact with the membrane at the cytosol - membrane interface/ cytosolic gydrophobic-hydrophilic environment.
b) the interaction is dependent on the ionic, metabolic stress and stain status of the cells.
c) In the dissociated state, the kihnetic constant like Km, Ki and Vmax of functional enzymes or affinity for ligands in case of enzymes or modulators is different than that in membrane bound state.
Accreted proteins are mainly the enzyme of glycolytic and redox pathway. They maintain the steady state of carbon flux and redox states for the generation of ATP by substrate level phosphorylation by altering the activity of key enzymes as reported in mild insulin dependent diabetes mellitus rat erythrocytes 25,26 . To maintain an optimum content, some of the enzymes particularly glyceraldehyde-3- phosphate dehydrogenase (G-3-PD) accrete with the membrane as observed on SDS-PAGE electrophoretogram 27 which is designated as band 6 proteins 17, while catalase accrete during oxidant stress. The accretion of G-3-PD and other enzymes is dependent on the ionic stength of the cytosol. Number of other funtional peptide accrete with the cytosolic segment of band 3 proteins. Accretion of cylindrin-a cylindrical acidic protein of unknown function and calmodulin are other examples. Cylindrin has been reported both in membrane and cytosolic fraction 28.29 while the erythrocyte calmodulin interacts with membrane via spectrin and Ca2+- ATPase cytosolic peptide.
Linker Proteins
Linker proteins link the extrinsic proteins with the intrinsic transmembrane proteins. These are extrinsic acylated (palmitoylated, myristolated) proteins having the following characteristics:
a) These proteins require ionic treatment only, such as high ionic strength/or strongly alkaline pH media devoid of nay detergent to dissociate them molecularly from the membrane.
b) in the dissociated stae, traces of either acylated or triphosphoinositoide moiety is present.
c) These proteins are soluble in neutral salt solution.
Ankyrin and band 4.1 are the major acylated linker proteins in erythrocyte membranes.
Ankyrin
Ankyrin link the specific segment of β spectrin subunit with major transmembrane glycoprotein i.e., band 3 It is a multiple phosphorylated asymmetric protein of 210 kDa in human erythrocyte membrane and is first protein to be identified to have a high-affinity binding site for spectrin on the inner surface of bilayer. Ankyrin has two distinet co-operative binding site for the erythrocyte anion exchanger 30 out of wihich, one site is salt sensitive which suggests that the interaction is mediated by electrostatic interaction, while the other site was not salt sensitive, indicating that the interaction is likely to be mediated by hydrogen bonding or hydrophobic contacts. Ankyrin is an acylated protein mainly having the palmitic acid residues at the band 3 binding segment31.
In terms of binomial numerology, it is known as band 2.1 and is susceptible to ca 2+ activated proteases. In pathophysiological and experimental states13 having high intracellular Ca2+ contents, different bands are observed between band 2 and 3 and are termed as band 2.1- 2.6. These bands are collectively known as syndeins 32. Reduction in ankyrin disrupts the linkage with the anion exchanger which destabilize the membrane associated with serve hemolytic anemia 33. Besides stablizing the membrane, the human erythrocyte ankyrin contain a 62kDa spectrin binding domain which is antigenically and structurally similar to P57, a membrane serine proteinase34.35.
Band 4.1 proteins
Protein 4.1 is composed of two identical 80 and 78 KDa peptide, the former differs from the later in carboxy terminal amino acid residues. Band 1.1 protein differs from ankyrin in term that it does not bind to a specific segment of one of the spectrin subunits, but instead requires the presence of both subunits to generate a stable complex comprising spectrin, band 4.1, actin, tropomyosin and band 4.9, it links the membranoskeleton with transmembrane glycophorin A as well as glycophorin C36 . The binding of band 4.1 with glycophorins is mediated by tri-phosphoinositoides 37. The altered band 4.1a/4.1b content observed on SDS_PAGE by using discontinuous buffer system of laemmli is explored by gerontologists to explain senescence of erythrocytes 38.
Amphitropic proteins
These proteins are linked to the membrane via some modified phospholipids constituents of the bilayer. One of the widely studied bilayer constituents is glycatedphosphatidyl inositol (GPI) having different moiety of carbohydrate attached to it in different animals 39. Some of the cell surface membrane proteins like ponticulin 11 that forms the major high affinity link between the plasma membrane and actin cortical network and Sn 23 protein12-a cell surface protein found on the human gut parasite S. manosi, were GPI anchored transmembrane proteins. The amphitropic proteins have the following characteristics:
a) These proteins can be dissociated from the bilayer either by the activation of a specific enzymes or high salt containing buffer system or by using ionic detergent.
b) They can acquire different conformers which depend on the constituents of the solution used to dissociate them from the bilayer.
c) The kinetic constant i.e. Vmax,km,Ki and affinity of enzymes differ when they exist as soluble form (i.e. dissociated state) from that of particulate form.
Erythrocyte membrane acetylcholinesterase (AChE), acid and alkaline phosphatases are the classical example of extracellular exofacial amphitropic proteins while different peptide constituent of functional G protein are example of cytosolic endofacial amphitropic proteins. Earlier AChE has been classified as an extrinsic as well as intrinsic proteins 40 which depends on the buffer system used to soublilize it. Different species of erythrocyte have different derivative of glycosylphosphatidyl inositol (GPI) due to which the response of bilayer to phosphoinositol phospholipase C(PI-PLC) to release AChE varies from species to species 41. The in-depth analysis of the sequence of event associated with the release of AChE due to PI-PLC activation will help us to understand precisely the biological significance of AChE in non-nucleated erythrocytes while acid and alkaline phosphatases cintent in serum is extensively used to evaluate the effect of toxicanst on different biological organization. The ubiquious occurrence of these proteins in biological system and their cellular and molecular biology has been extensively reviewed 42. The relevance of these proteins in evolution has also been elaborated43.
Structural transmembrane proteins
The structural tranmembrane proteins are integral membrane proteins generally having a single, 22-25 amino acid residues containing hydrophobic domain. The characteristic feature of these proteins are:
a) They require drastic treatment with reagents such as detergens, bile acids, protein denaturanst or organic solvent to dissociate them from hydrophobic domain of biomembranes.
b) in many instances the functional proteins remain associated with lipids when isolated.
c) If completely dissociated from lipids, they are usually highly insoluble or aggregated in neutral aqueous buffers thus having an altered cooperatively with its ligands.
Different glycophorins are major structural transmembrane proteins in erythrocyte membrane. The glycoproteins constituting transmembrane proteins differ in the length and complexity of the oligosaccharide chains and the proportions of sialic acid residues attached to them . These are detected by PAS staining of the SDS-PAGE gels. The glycoproteins are characterized on the basis of amino acid carbohydrate and sialic acid residues into glycophorin A(GPA), glycophorin B(GPB) , glycophorin C (GPC) and glycophorin D(GPD)44. Glycophorin A is a also known as PASI, GPB as PASIII while GPC as PASII . These glycophorins contain a single membrane spanning segment of 22-23 uncharged amino acids just-long enough to cross the non-polar region of the bilayer having N terminal on the extracellular side Glycophorin A is the carrier of MN blood group while glycophorin B is the carrier of Ss blood group. Glycophorin C consists different variant of β sialoglycopeptides while glycophorin D contains α sialoglycopeptides only. On the contrary, the ABO, Rh and lewis blood group of human are determined by glycosphingolipids. Glycophorin C and D is responsible for leech phenotype lacking the common Gebrich blood group. In the elliptocytes of homozygous Gerbich blood group dieficient individual, the relative lack of linker protein 4.1 has also been observed 45. The analysis of these mutant erythrocyte membrane organization supported that glycophorin C possesses the binding site for protein 4.1 and tus is essential fro the maintenance of discocyte shape.
Functional transmembrane proteins
The functional transmembrane proteins are either multimeric complex or oligomeric integral proteins generally having 45-220 amino-acid residues containning 2-7 hydrophobic domain spanning segment each having 22-25 amino acid residues. The characteristic feature of these protein are;
a) They require drastic treatment with reagents such as detergents, bile acids, proteins denaturants or organic solvent to dissociate them from hydrophobic domain of biomembranes.
b) In many instances, the functional proteins exist in the form of protein phospholipid particulate complex.
c) If ciompletely dissociated from lipids, they are usually highly insoluble or aggregated in neutral aqueous buffers thus having an altered cooperativity with its ligands or are rendered functionless due to the loss of their capability to phosphorylate their specific amino-acid residues.
The cation dependent ATPases, anion and glucose transporters exists in the form of protein phospholipids particulate compleses and are generally made up of multimers of subunits occasionally capable of forming transmembrane channels. The cation dependent ATPases and transporters maintain the optimum contents of osmotically and physiologically necessary ionic species.
Anion and glucose transporter
The heterogeneous protein of about 90.0 kDa is involved in the exchange of chloride, bicarbonate and water across the red cell membranes. It is constituted by a group of heterogeneous polypeptides known as band 3 protein . Band 3 is glycosylated in extracellular surface while the cytosolic domain possesses the binding site of haemoglobin, glycolytic enzymes 46 and the linker protein i.e. ankyrin. The site of band 3 polypeptides involved in binding with ankyrin (band 2.1) maintains the structural integrity of erythrocytes by linking membranoskeleton with the hydrophobic domain . Band 3 protein is also the precursor of 62 kDa senescent autontigen 47 as well as band 4.5 the glucose tranporter 48. It is believed that the senescent autoantigen and glucose tranporter are produced by the proteolytic degradation of anion transporter proteins. The anion transporter proteins have multiple polypeptide chains across the lipid bilayer. These polypeptides have charged amino acids. The side chain of specific amino acid pair with specific amino acids to form aqueous channel across the lipid bilayer . As these aqueous channels are involved in the transfer of anion, the polypeptides constituting it are called anion channel proteins (ACP) . The ACP is main component of band 3 and is made up of two distinct segments: i) a membrane spanning segment, facilitating the exchange of anion across the membranes; ii) a cytoplasmic segment which is the locus of multiple interactions between the integral domain of the proteins, the cytoskeleton and cytosolic proteins.
The functional ACP is a phosphoprotein phosphorylated by caseinkinases ands band 3 tyrosine kinase. Vasseur49 reoorted that when band 3 is phosphorylated in the presence of Mg24 51% of phosphorylated amine acid is tyrosine,while phosphorylated serine and phosphotheorinine are 34% and 15% respectively. Phosphorylation of the cytoplasmic segment regulates the interaction of ACP with cytosolic structural and functional proteins.
The other band 3 proteins are glycophorin A having a single polypeptide of about 22-23 neutral amino-acids in membrane spanning segment while Ca 2+ ATPase is another protein. All the proteins of band 3 are glycosylated to varying degree and undrgo phosphorylation to execute their function. Cation dependent ATPases are elaborately discussed and described earlier7. Although, erythroid insulin receptors and many other receptors of hormones, growth factors, cytokinins etc. have been characterized. The receptors and many other receptors of hormones, growth factors, cytokinins etc. have been characterized. The receptors of other ligands present in extra-cellular hydrophilic environment of erythrocyte membrane along with the enzymatic mechanism of isoprenylation 50 and accretion of different G peptides 51 at cytosolic hyrophobic/hydrophilic interface need to be specifically analyzed and understood. After extensive structural and functional analysis and the subjective and intuitive understanding of the localization of receptors and various G peptide in varied dynamic state, and attempt should be made to classify the above mentioned proteins to any of the six classes proposed in physio-chemical classification of membrane proteins.
In different non-erythroid cell diverse type of proteins which are functionally similar to that of erythroid cell have been reported and extensively reviewed in case of erythroid spectrin and non-erythroid fordin 19.52.53 Thus, the criteria proposed above to group membrane proteins into six classes may be extensively used for classifying membrane proteins isolated from different cell and organs. Unanimous acceptance of above classification may provide impetus to develop biomembranology as a separate multidisciplinary discipline of basic and applied biological sciences.
Acknowledgement
Author express his gratitude to Prof. J,S. Rathore and Prof. M. Suhail for constant encouragement and to the Principal Trustee, Shri Gupta Institute, Jabalpur, for enduring financial support.
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Dinesh kumar Gupta
Shree Gupta Institute, 903, Bai-ka-Bagicha,
jabalpur(M.P.)- 482001, India
Abstract
On the basis of shape/structure, function performed and localization in fluid matrix, the membrane protein are classified into six classes. Different proteins of well characterized erythrocyte membranes are classified under proposed physio-chemial classification. As there exists phylogenicially and structurally similar proteins in wide diversity of biomembranes, it is needed to develop a general classification of membrane proteins.
Key words: Biomembranes, amphitropic proteins, accreted proteins, linker proteins, modulator proteins, cytosolic functional proteins.
Introduction
Traditionally, membrane proteins were classified into intrinsic and extrinsic proteins. As new proteins were identified, terms like lipid anchored protein, acylated and (iso) prenylated proteins were used. Intrinsic proteins were later on classified into type I,II,III & IV while acylated proteins are classified as type V. The transmembrane GPI anchored proteins (dually attached ) were classified as type VI proteins . In the present classification of membrane proteins, these are classified into six classes. The different classes of membrane proteins are cytosolic structural proteins, cytosolic functional proteins, linker proteins, amphitropic proteins, structural and functional transmembrane proteins. The criteria of classification is illustrated by quoting the example of erythrocyte membrane proteins, As there exists proteins similar to that of erythrocyte in different non-erythroid cells, this classification can be accepted as general classification of membrane proteins.
Dynamic state of membrane is due to the presence of diverse types of protein molecules. Proteins exists in different configuration by acquiring a wide variety of conformation. The globular conformation dominates the expanded conformation and is interdigitated between the hydrophobic domain of lipid bilayer by means of hydrophobic interaction or by means of covalent bonds1. On freeze fracture microscopy, some of these proteins exists as intra-membranous particles2. These globular intra-membranous particles are important functional complexes of dynamic biomembrane and are involved in the transport of osmolytes, solutes, metabolites and targeting of peptides, as well as proper orientation of receptor and adaptor molecules. Contrary to globur proteins, the expanded conformations possessing macromolecules form the immunogenic domain, membranoskeleton, cytoskeleton3 etc. and are important macromolecular complexes in discriminating between self and non-self, cellular shape and biomembrane macromolecular architecture in the three dimensional spatiotemporal frame - work.
Existing system of classification
On the basis of solubility of membrane proteins i.e. the type of solution required to disintegrate and extract proteins from their hydrophobic domain, the membrane proteins were traditionally classified as extrinsic (peripheral) and intrinsic(integral) proteins4. In 1981, Houslay had classified membrane proteins into six classes out of which class I - to class IV were integral protein and class V and class VI were peripheral proteins. Class I includes transmembrane globular integral proteins, class II transmembrane fibrous integral protein, class I I I globular protein in aqueous phase but anchored by an hydrophilic pedicle that does not span the bilayer while that of class IV includes globular integral proteins associated essentially with one or other half of the bilayer. In Houslay system of classification, class V were peripheral proteins, associated essentially through interaction with phospholipids while class VI were peripheral proteins capable of binding to specific integral transmembrane proteins. The above system of nomenclature does not gain prominence among membranologists as it had given more emphasis on the physico-chemical properties of the protein rather than to the range of functions that a particular protein performs in the functional membrane. Ramasarma6 used the term lipid anchored proteins to all those proteins which were covalently attached with the constituent of hydrophobic domain of membrane by forming covalent linkage with the hydrophobic chains of fatty acids. Gupta7 while reviewing membrane proteins have classified it into six classes on the basis of (a) the function performed by protein to maintain dynamic state of membrane and (b) the spatial localization of protein in the three dimensional hydrophobic domain. Singer8 has further classified single hydrophobic domain containing integral protein as type I and II while proteins possessing multiple hydrophobic domain as type III and IV . The acylated and GPI anchored protein were termed as type V9 and transmembrane GPI anchored proteins as type VI10 . These membrane anchored transmembrane proteins are the cell surface protein 'ponticulin'11 and cysteine rich hydrophobic Sm 23 protein, a surface protein of human parasite Schistosoma mansoni 12 . Extensive analysis of erythroid and noneythroid membrance proteins of normal, pathological and different physiological state as well as in vivo and in vitro manipulation technuques 13 have generated diverse range of data and is expected to generate more information on some unexplored physio-chemical nature of these proteins and macromolecular complexes. Thus, the existing system of classification needs to be modified and elaborated.
Tenets and concept of physio-chemical classification
To achieve the above task, the basic tenet proposed for physio-chemical classification of membrane protein is that, the range of functional and structural niche occupied by protein is not only dependent on the physico-chemical sturcture of protein but also on the localization of proteins in the spatio-temporal frame work of hydrophobic domain of membrane existing in the dynamic state. This is well illustrated by the fact that the isoprenylation(farnesylation, geranyl-geranylation) of g protein 14 (modulator component of modulator subsysem15) and other ras related protein is a critical step for sub-cellular localization and acquisition of biological activities of the cell growth and regulation. The other useful concept is the existence of three heterogenous domains in the functional membrane which were termed as extracellular hydrophilic environment, lipophilic-hydrophobic environment and cytosolic-hydrophilic environment 15. The above mentioned physio-chemical tenets and the concept of existence of heterogeneous domain in a functional biomembrane are the basis of the physio-chemical classification of the membrane proteins.
Physio-chemical classes of membrane proteins
Erythrocyte membrane proteins are classified into six classes on the basis of range of functions performed by the proteins to maintain dynamic state of erythrocyte, as well as spatial localization of the proteins as observed by different techniques, viz., microscopic 16, electrophoretic 17 and immunogenic labeling analysis. The six classes of erythrocyte membrane proteins are i) cytosolic structural proteins ii) cytosolic functional proteins iii) linker proteins, iv) amphitropic proteins, v) structural transmembrane proteins and vi) functional transmembrane proteins.
Table-1 summarizes the proposed classification of well characterized erythrocyte membrane proteins. The criteria used to classify membrane pr4oteins into six classes are elaborated by illustrating the example of erythrocyte membrane proteins or their macromolecular content.
No. Classes Erythroid Proteins Physio-chemical characteristics
1 Cytosolic structural proteins Spectrin α Soluble in neutral aqueous buffer as they are totally free from phospholipids and can be solubilized by altering ionic strength of the medium or by adding chelating agents.
Spectrin β
Actin gelatinous
Actin filamentous
2 Cytosolic functional proteins Glyceraldehyde-3-phosphate,dehydrogenase, catalase, membrane bound hexokinase & other enzymes Gets accreted with the hydrophobic domain in presence of ionic species or generation of metabolite and redox potential during different physiological and pathophysiological state.
3 Linker proteins Ankyrin, Band 4.1 Soluble in netural salt solution and generally exist as an acylated form can be solubilized from the membrane by altering ionic strength and pH of the medium.
4 Amphitropic proteins Acetylcholinesterases The functional proteins whose characteristic anre altered i.e. affinity for substrate or Kd when solubilized from the membrane by the enzymatic action, high salt or ionic detergent containing buffer.
Acid and alkaline phosphatases
Peptides of G proteins
5 Structural transmembrane proteins Glycophorin C The single hydrophobic domain containing integral peptides generally remain associated with phospholipids when extracted from membrane.
6 Functional transmembrane proteins Anion transporters, Cation dependent ATPases, Receptors of different ligands These are generally multimeric hydrophobic domain containing intergrl proteins when extracted exist as protein-phospholipid particulate complexes and are dependent on a particular hydrophobic requirement to execute their function.
Cytosolic structural proteins:
These proteins are extrinsic membrane proteins18 having the following characteristics:
a) They require only mild treatment such as the alteration in the ionic strength of the medium or the addition of a chelating agent to dissociate them moleculary from the membranes:
b) They dissociate totally from lipids, and
c) In the dissociated state they are relatively soluble in neutral aqueous buffers.
Spectrin and actin come under this group. Spectrin is the most abundant cytosolic structural protein in erythroid and non-erythroid cells. Human erythrocytes membrane spectrin is composed of 240 kDa alpha (α or band 1) and 225 kDa beta(β or band 2) peptide subunits. The molecular diversity and evolution of erythroid and non-erythroid spectrin had already been elaborated 19.20 . These a and b subunits can form homodiamer i.e. (α)2 and (β)2 in pathological states, heterodimers i.e. (αβ ) and tetramer(αβ) 2 in normal physiological state, while other oligomers are also observed in solutions. In heterodimer, the amino terminal of β subunit aligned with carboxy terminal of α subunit parallel to one another. These dimers of spectrin self associate in the region containing 80 kDa segment that begins at the amino terminal of the α subunit . Besides self associating segment, human and other animal erythrocyte membrane spectrin contains specific binding sites for ankyrin, actin, band 4.1, calmodulin 21 and the metabolite 2.3. diphosphoglycerate 22-24 . The subunits of spectrin forming the head group have ankyrin binding segment, while tail of α/ β spectrin dimer, actin and band 4.1 binds and forms the meshwork of protein. Actin exists in two form i.e. G actin and F actin. The polymerized and filamentous form of F actin is the constituents of membranoskeleton junctional complexes. The major protein constituents of membranoskeletal complex are tail region of α/ β spectrin dimer, band 4.1 and actin while myosin, tropomyosin and band 4.9 are the minor component stablizing the F actin -F actin, actin-spectrin interaction.
Cytosolic functional proteins.
Cytosolic functional proteins partition themselves between the cytosol membrane interface (cytosolic hydrophilic/hydrophobic environement ) in order to maintain an optimum state. These are membrane accreted proteins having following characteristics:
a) These proteins interact with the membrane at the cytosol - membrane interface/ cytosolic gydrophobic-hydrophilic environment.
b) the interaction is dependent on the ionic, metabolic stress and stain status of the cells.
c) In the dissociated state, the kihnetic constant like Km, Ki and Vmax of functional enzymes or affinity for ligands in case of enzymes or modulators is different than that in membrane bound state.
Accreted proteins are mainly the enzyme of glycolytic and redox pathway. They maintain the steady state of carbon flux and redox states for the generation of ATP by substrate level phosphorylation by altering the activity of key enzymes as reported in mild insulin dependent diabetes mellitus rat erythrocytes 25,26 . To maintain an optimum content, some of the enzymes particularly glyceraldehyde-3- phosphate dehydrogenase (G-3-PD) accrete with the membrane as observed on SDS-PAGE electrophoretogram 27 which is designated as band 6 proteins 17, while catalase accrete during oxidant stress. The accretion of G-3-PD and other enzymes is dependent on the ionic stength of the cytosol. Number of other funtional peptide accrete with the cytosolic segment of band 3 proteins. Accretion of cylindrin-a cylindrical acidic protein of unknown function and calmodulin are other examples. Cylindrin has been reported both in membrane and cytosolic fraction 28.29 while the erythrocyte calmodulin interacts with membrane via spectrin and Ca2+- ATPase cytosolic peptide.
Linker Proteins
Linker proteins link the extrinsic proteins with the intrinsic transmembrane proteins. These are extrinsic acylated (palmitoylated, myristolated) proteins having the following characteristics:
a) These proteins require ionic treatment only, such as high ionic strength/or strongly alkaline pH media devoid of nay detergent to dissociate them molecularly from the membrane.
b) in the dissociated stae, traces of either acylated or triphosphoinositoide moiety is present.
c) These proteins are soluble in neutral salt solution.
Ankyrin and band 4.1 are the major acylated linker proteins in erythrocyte membranes.
Ankyrin
Ankyrin link the specific segment of β spectrin subunit with major transmembrane glycoprotein i.e., band 3 It is a multiple phosphorylated asymmetric protein of 210 kDa in human erythrocyte membrane and is first protein to be identified to have a high-affinity binding site for spectrin on the inner surface of bilayer. Ankyrin has two distinet co-operative binding site for the erythrocyte anion exchanger 30 out of wihich, one site is salt sensitive which suggests that the interaction is mediated by electrostatic interaction, while the other site was not salt sensitive, indicating that the interaction is likely to be mediated by hydrogen bonding or hydrophobic contacts. Ankyrin is an acylated protein mainly having the palmitic acid residues at the band 3 binding segment31.
In terms of binomial numerology, it is known as band 2.1 and is susceptible to ca 2+ activated proteases. In pathophysiological and experimental states13 having high intracellular Ca2+ contents, different bands are observed between band 2 and 3 and are termed as band 2.1- 2.6. These bands are collectively known as syndeins 32. Reduction in ankyrin disrupts the linkage with the anion exchanger which destabilize the membrane associated with serve hemolytic anemia 33. Besides stablizing the membrane, the human erythrocyte ankyrin contain a 62kDa spectrin binding domain which is antigenically and structurally similar to P57, a membrane serine proteinase34.35.
Band 4.1 proteins
Protein 4.1 is composed of two identical 80 and 78 KDa peptide, the former differs from the later in carboxy terminal amino acid residues. Band 1.1 protein differs from ankyrin in term that it does not bind to a specific segment of one of the spectrin subunits, but instead requires the presence of both subunits to generate a stable complex comprising spectrin, band 4.1, actin, tropomyosin and band 4.9, it links the membranoskeleton with transmembrane glycophorin A as well as glycophorin C36 . The binding of band 4.1 with glycophorins is mediated by tri-phosphoinositoides 37. The altered band 4.1a/4.1b content observed on SDS_PAGE by using discontinuous buffer system of laemmli is explored by gerontologists to explain senescence of erythrocytes 38.
Amphitropic proteins
These proteins are linked to the membrane via some modified phospholipids constituents of the bilayer. One of the widely studied bilayer constituents is glycatedphosphatidyl inositol (GPI) having different moiety of carbohydrate attached to it in different animals 39. Some of the cell surface membrane proteins like ponticulin 11 that forms the major high affinity link between the plasma membrane and actin cortical network and Sn 23 protein12-a cell surface protein found on the human gut parasite S. manosi, were GPI anchored transmembrane proteins. The amphitropic proteins have the following characteristics:
a) These proteins can be dissociated from the bilayer either by the activation of a specific enzymes or high salt containing buffer system or by using ionic detergent.
b) They can acquire different conformers which depend on the constituents of the solution used to dissociate them from the bilayer.
c) The kinetic constant i.e. Vmax,km,Ki and affinity of enzymes differ when they exist as soluble form (i.e. dissociated state) from that of particulate form.
Erythrocyte membrane acetylcholinesterase (AChE), acid and alkaline phosphatases are the classical example of extracellular exofacial amphitropic proteins while different peptide constituent of functional G protein are example of cytosolic endofacial amphitropic proteins. Earlier AChE has been classified as an extrinsic as well as intrinsic proteins 40 which depends on the buffer system used to soublilize it. Different species of erythrocyte have different derivative of glycosylphosphatidyl inositol (GPI) due to which the response of bilayer to phosphoinositol phospholipase C(PI-PLC) to release AChE varies from species to species 41. The in-depth analysis of the sequence of event associated with the release of AChE due to PI-PLC activation will help us to understand precisely the biological significance of AChE in non-nucleated erythrocytes while acid and alkaline phosphatases cintent in serum is extensively used to evaluate the effect of toxicanst on different biological organization. The ubiquious occurrence of these proteins in biological system and their cellular and molecular biology has been extensively reviewed 42. The relevance of these proteins in evolution has also been elaborated43.
Structural transmembrane proteins
The structural tranmembrane proteins are integral membrane proteins generally having a single, 22-25 amino acid residues containing hydrophobic domain. The characteristic feature of these proteins are:
a) They require drastic treatment with reagents such as detergens, bile acids, protein denaturanst or organic solvent to dissociate them from hydrophobic domain of biomembranes.
b) in many instances the functional proteins remain associated with lipids when isolated.
c) If completely dissociated from lipids, they are usually highly insoluble or aggregated in neutral aqueous buffers thus having an altered cooperatively with its ligands.
Different glycophorins are major structural transmembrane proteins in erythrocyte membrane. The glycoproteins constituting transmembrane proteins differ in the length and complexity of the oligosaccharide chains and the proportions of sialic acid residues attached to them . These are detected by PAS staining of the SDS-PAGE gels. The glycoproteins are characterized on the basis of amino acid carbohydrate and sialic acid residues into glycophorin A(GPA), glycophorin B(GPB) , glycophorin C (GPC) and glycophorin D(GPD)44. Glycophorin A is a also known as PASI, GPB as PASIII while GPC as PASII . These glycophorins contain a single membrane spanning segment of 22-23 uncharged amino acids just-long enough to cross the non-polar region of the bilayer having N terminal on the extracellular side Glycophorin A is the carrier of MN blood group while glycophorin B is the carrier of Ss blood group. Glycophorin C consists different variant of β sialoglycopeptides while glycophorin D contains α sialoglycopeptides only. On the contrary, the ABO, Rh and lewis blood group of human are determined by glycosphingolipids. Glycophorin C and D is responsible for leech phenotype lacking the common Gebrich blood group. In the elliptocytes of homozygous Gerbich blood group dieficient individual, the relative lack of linker protein 4.1 has also been observed 45. The analysis of these mutant erythrocyte membrane organization supported that glycophorin C possesses the binding site for protein 4.1 and tus is essential fro the maintenance of discocyte shape.
Functional transmembrane proteins
The functional transmembrane proteins are either multimeric complex or oligomeric integral proteins generally having 45-220 amino-acid residues containning 2-7 hydrophobic domain spanning segment each having 22-25 amino acid residues. The characteristic feature of these protein are;
a) They require drastic treatment with reagents such as detergents, bile acids, proteins denaturants or organic solvent to dissociate them from hydrophobic domain of biomembranes.
b) In many instances, the functional proteins exist in the form of protein phospholipid particulate complex.
c) If ciompletely dissociated from lipids, they are usually highly insoluble or aggregated in neutral aqueous buffers thus having an altered cooperativity with its ligands or are rendered functionless due to the loss of their capability to phosphorylate their specific amino-acid residues.
The cation dependent ATPases, anion and glucose transporters exists in the form of protein phospholipids particulate compleses and are generally made up of multimers of subunits occasionally capable of forming transmembrane channels. The cation dependent ATPases and transporters maintain the optimum contents of osmotically and physiologically necessary ionic species.
Anion and glucose transporter
The heterogeneous protein of about 90.0 kDa is involved in the exchange of chloride, bicarbonate and water across the red cell membranes. It is constituted by a group of heterogeneous polypeptides known as band 3 protein . Band 3 is glycosylated in extracellular surface while the cytosolic domain possesses the binding site of haemoglobin, glycolytic enzymes 46 and the linker protein i.e. ankyrin. The site of band 3 polypeptides involved in binding with ankyrin (band 2.1) maintains the structural integrity of erythrocytes by linking membranoskeleton with the hydrophobic domain . Band 3 protein is also the precursor of 62 kDa senescent autontigen 47 as well as band 4.5 the glucose tranporter 48. It is believed that the senescent autoantigen and glucose tranporter are produced by the proteolytic degradation of anion transporter proteins. The anion transporter proteins have multiple polypeptide chains across the lipid bilayer. These polypeptides have charged amino acids. The side chain of specific amino acid pair with specific amino acids to form aqueous channel across the lipid bilayer . As these aqueous channels are involved in the transfer of anion, the polypeptides constituting it are called anion channel proteins (ACP) . The ACP is main component of band 3 and is made up of two distinct segments: i) a membrane spanning segment, facilitating the exchange of anion across the membranes; ii) a cytoplasmic segment which is the locus of multiple interactions between the integral domain of the proteins, the cytoskeleton and cytosolic proteins.
The functional ACP is a phosphoprotein phosphorylated by caseinkinases ands band 3 tyrosine kinase. Vasseur49 reoorted that when band 3 is phosphorylated in the presence of Mg24 51% of phosphorylated amine acid is tyrosine,while phosphorylated serine and phosphotheorinine are 34% and 15% respectively. Phosphorylation of the cytoplasmic segment regulates the interaction of ACP with cytosolic structural and functional proteins.
The other band 3 proteins are glycophorin A having a single polypeptide of about 22-23 neutral amino-acids in membrane spanning segment while Ca 2+ ATPase is another protein. All the proteins of band 3 are glycosylated to varying degree and undrgo phosphorylation to execute their function. Cation dependent ATPases are elaborately discussed and described earlier7. Although, erythroid insulin receptors and many other receptors of hormones, growth factors, cytokinins etc. have been characterized. The receptors and many other receptors of hormones, growth factors, cytokinins etc. have been characterized. The receptors of other ligands present in extra-cellular hydrophilic environment of erythrocyte membrane along with the enzymatic mechanism of isoprenylation 50 and accretion of different G peptides 51 at cytosolic hyrophobic/hydrophilic interface need to be specifically analyzed and understood. After extensive structural and functional analysis and the subjective and intuitive understanding of the localization of receptors and various G peptide in varied dynamic state, and attempt should be made to classify the above mentioned proteins to any of the six classes proposed in physio-chemical classification of membrane proteins.
In different non-erythroid cell diverse type of proteins which are functionally similar to that of erythroid cell have been reported and extensively reviewed in case of erythroid spectrin and non-erythroid fordin 19.52.53 Thus, the criteria proposed above to group membrane proteins into six classes may be extensively used for classifying membrane proteins isolated from different cell and organs. Unanimous acceptance of above classification may provide impetus to develop biomembranology as a separate multidisciplinary discipline of basic and applied biological sciences.
Acknowledgement
Author express his gratitude to Prof. J,S. Rathore and Prof. M. Suhail for constant encouragement and to the Principal Trustee, Shri Gupta Institute, Jabalpur, for enduring financial support.
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