Amino acid sequence of an alpha-delta-glycophorin hybrid. A structure reciprocal to Sta delta-alpha-glycophorin hybrid

In this report we examine the primary sequence of a variant glycophorin obtained from erythrocytes of an individual who exhibits an unusual MNSs blood group phenotype. We show that this protein is a hybrid molecule constructed from sequences of alpha- and delta-glycophorins (glycophorins A and B) in a alpha-delta arrangement. Serological typing revealed that the donor's phenotype was M+N+S+s+U+; yet his erythrocytes reacted with some but not all examples of anti-S antisera. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a variant glycophorin band, and immunoblotting and reaction with N-glycanase suggested that its amino terminus resembled that of M-alpha-glycophorin but that its carboxyl terminus did not. A preparation highly enriched in the variant was obtained and used to generate peptide fragments for sequencing. The sequence revealed that the variant was a hybrid molecule whose amino terminus corresponded to M-alpha-glycophorin and whose carboxyl terminus corresponded to S-delta-glycophorin. CNBr cleavage of the variant glycophorin yielded four peptides. The sequence of the amino-terminal CNBr peptide (residues 1-8) was identical to the amino-terminal octapeptide of M-alpha-glycophorin. The proceeding peptide (residues 9-61) contained a segment identical to residues 9-58 of alpha glycophorin, but its carboxyl-terminal sequence had the Gly-Glu-Met sequence from S-delta-glycophorin (residues 27-29). The other two peptides, insoluble in aqueous solutions, contained highly hydrophobic sequences, identical to residues 30-52 and 53-68 of delta-glycophorin. Sequences of overlapping peptides generated by trypsin and V8 protease confirmed the hybrid nature of the variant glycophorin: residues 1-58 were identical to residues 1-58 of M-alpha-glycophorin, and residues 59-100 were entirely identical to residues 27-68 of S-delta-glycophorin. The variant glycophorin is expected to have 4 additional residues at its carboxyl terminus that correspond to the carboxyl-terminal residues 69-72 of delta-glycophorin. The amino acid sequence arrangement of the variant alpha-delta-glycophorin is an exact reciprocal of that found in another hybrid glycophorin, Sta, that is a delta-alpha hybrid. We propose that the two hybrid glycophorins represent the two possible products resulting from a reciprocal recombination event.     

A re-examination of the effects of chymotrypsin and trypsin on the erythrocyte membrane surface topology

Silver/Coomassie blue staining of human erythrocyte membrane electrophoretograms permits simultaneous visualization and color differentiation of asialoproteins, sialoglycoproteins and lipids in the same gel. Using this technique evidence is provided that chymotrypsin cleaves glycophorin A as well as band 3. The chymotryptic fragmentation pattern of glycophorin A in situ intact cells was different from that generated by trypsin treatment. Chymotryptic cleavage of band 3 generated two Coomassie blue stained fragments at 62,000 and 38,000 Mr, whereas simultaneous cleavage of glycophorin A dimer and glycophorin A B heterodimer yielded yellow silver stained fragments at 68,000 and 47,000 Mr. Trypsin cleaved glycophorin A dimer (88,000 Mr) and monomer (38,000 Mr) to form membrane associated fragments of Mr = 40,000 and 18,000 respectively.     

Heterogeneous post-translational modification of Actinobacillus actinomycetemcomitans fimbrillin

Fresh isolates of Actinobacillus actinomycetemcomitans produce bundle-forming fimbriae. The exact molecular mass of A. actinomycetemcomitans fimbrillin, a structural subunit of fimbriae, was determined by liquid chromatography-electrospray ionization mass spectrometry. Three major molecular species with 6,226.0, 6,366.0, and 6,513.0 Da were detected in a purified fimbrial fraction from the strain 310-a. These molecular masses were significantly higher than the molecular weight (5,118 Da) calculated from nucleotide sequence data of the fimbrillin gene, flp, suggesting that the fimbrial peptides were post-translationally modified. Modification of the fimbrial peptides was also suggested by an N-terminal amino acid sequence analysis of fimbrillin peptic fragments, with the modified amino acids being due to seven serine or asparagine residues located in the C-terminal region. A periodate oxidation/biotin-hydrazide labeling assay of fimbrillin suggested that it might be glycosylated.     

Detection of oxidized lipid-modified erythrocyte membrane proteins by radiolabeling with tritiated borohydride

Human erythrocyte ghosts treated with tert-butyl hydroperoxide or ADP-Fe3+ incorporated radioactivity on reduction with tritiated borohydride. The tritium incorporation closely correlated with membrane lipid oxidation as assessed by the formation of thiobarbituric acid-reactive substances and fluorescent substances. Treatment of ghosts with the inducers in the presence of butylated hydroxytoluene, thiourea, or desferrioxamine suppressed the tritium incorporation in the subsequent reduction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the tritiated ghost proteins showed that the label was incorporated into the intermolecularly cross-linked and the uncross-linked proteins of bands 1, 2, 3, 4.1, 4.2, 5 and 6, and into the noncross-linked glycophorin A (PAS-1). Glycophorin A was hardly cross-linkable but modified during membrane lipid oxidation. Possible candidates for producing borohydride-reducible functions in the proteins are various mono- and bifunctional aldehydes, as well as those for producing fluorescence and cross-links. A part of thiobarbituric acid-reactive or fluorescent substances may be involved in borohydride reduction and tritium labeling.     

Orientation of the glucose transporter in the human erythrocyte membrane. Investigation by in situ proteolytic dissection

Glucose transporter proteins (zone 4.5) which had been photoaffinity labeled with [3H]cytochalasin B in human erythrocyte ghosts were subjected to enzymatic dissection in order to study the transmembrane disposition of the protein in situ. Proteolytic enzymes as well as glycosidases were used to treat unsealed and resealed ghosts in order to explore the various membrane domains of the transporter in a topographically defined manner. Limited digestion of sealed ghosts with trypsin had no effect on the apparent Mr of the transporter (55,000). Similar treatment in unsealed ghosts, however, resulted in the generation of a major fragment of 21.5 kDa, along with several minor fragments. Thermolysin also had no effect on sealed ghosts but caused a complete loss of radiolabel from the zone 4.5 region with no lower-molecular-weight fragments being retained on the gel. Chymotrypsin treatment resulted in the generation of a single peak, Mr = 18,400, in both sealed and unsealed ghosts indicating its action occurs at the outer surface. Digestion with carboxypeptidase and aminopeptidase indicate the C-terminal end of the transporter is located exterior to the membrane with the N terminus located at the cytoplasmic surface. Treatment with endoglycosidase resulted in a shift of mobility of the transporter to a lower Mr of 49,000. The results obtained indicate that the carbohydrate is located near the C-terminal end and that the cytochalasin B-binding site is located near the cytoplasmic N-terminal end.     

Intermolecular disulfide bonds link specific high-molecular-weight glutenin subunits in wheat endosperm.

A detergent wash extracted soluble proteins from wheat flour, leaving a residue enriched with insoluble glutenin aggregates. Digestion of this residue with endoproteinase Lys-C, which showed a limited specificity for glutenin subunits, produced several peptides with apparent molecular weights close to those of intact high-molecular-weight glutenin subunits. N-terminal sequencing indicated that the isolated peptides were composed of high-molecular-weight glutenin subunit fragments joined by an intermolecular disulfide bond. In two of these peptides, only two components were found, one from an x-type subunit and the other from a y-type subunit. The isolated peptides all contained at least one x-type C-terminal region and one y-type N-terminal region, suggesting a specific orientation to the intermolecular disulfide linkage.     

PAS domain of the Aer redox sensor requires C-terminal residues for native-fold formation and flavin adenine dinucleotide binding

The Aer protein in Escherichia coli is a membrane-bound, FAD-containing aerotaxis and energy sensor that putatively monitors the redox state of the electron transport system. Binding of FAD to Aer requires the N-terminal PAS domain and residues in the F1 region and C-terminal HAMP domain. The PAS domains of other PAS proteins are soluble in water. To investigate properties of the PAS domain, we subcloned segments of the aer gene from E. coli that encode the PAS domain with and without His6 tags and expressed the PAS peptides in E. coli. The 20-kDa His6-Aer2-166 PAS-F1 fragment was purified as an 800-kDa complex by gel filtration chromatography, and the associating protein was identified by N-terminal sequencing as the chaperone protein GroEL. None of the N-terminal fragments of Aer found in the soluble fraction was released from GroEL, suggesting that these peptides do not fold correctly in an aqueous environment and require a motif external to the PAS domain for proper folding. Consistent with this model, peptide fragments that included the membrane binding region and part (Aer2-231) or all (Aer2-285) of the HAMP domain inserted into the membrane, indicating that they were released by GroEL. Aer2-285, but not Aer2-231, bound FAD, confirming the requirement for the HAMP domain in stabilizing FAD binding. The results raise an interesting possibility that residues outside the PAS domain that are required for FAD binding are essential for formation of the PAS native fold.     

Gating of a voltage-dependent channel (colicin E1) in planar lipid bilayers: translocation of regions outside the channel-forming domain

Summary C-terminal fragments of colicin E1, ranging in mol wt from 14.5 to 20kD, form channels with voltage dependence and ion selectivity qualitatively similar to those of whole E1, placing an upper limit on the channel-forming domain. Under certain conditions, however, the gating kinetics and ion selectivity of channels formed by these different E1 peptides can be distinguished. The differences in channel behavior appear to be correlated with peptide length. Enzymatic digestion with trypsin of membrane-bound E1 peptides converts channel behavior of longer peptides to that characteristic of channels formed by shorter fragments. Apparently trypsin removes segments of protein N-terminal to the channel-forming region, since gating behavior of the shortest fragment is little affected by the enzyme. The success of this conversion depends on the side of the membrane to which trypsin is added and on the state, open or closed, of the channel. Trypsin modifies only closed channels from thecis side (the side to which protein has been added) and only open channels from thetrans side. These results suggest that regions outside the channel-forming domain affect ion selectivity and gating, and they also provide evidence that large protein segments outside the channel-forming domain are translocated across the membrane with channel gating.     

Appearance of elongation factor Tu in the outer membrane of sucrose-dependent spectinomycin-resistant mutants of Escherichia coli

When sucrose-dependent spectinomycin-resistant (Sucd-Spcr) mutants of Escherichia coli were grown in the absence of sucrose, a new protein appeared in the membrane fraction insoluble in Triton X-100. The protein had a hydrophobic nature. However, unlike other outer membrane proteins the new protein was extracted with sodium dodecyl sarcosinate. The new protein was found to be identical with elongation factor Tu (EF-Tu), as judged from the electrophoretic mobility in three different gel systems, coprecipitation with the antiserum against EF-Tu, the profiles of peptide fragments produced with three different proteases and analyses of N-terminal and C-terminal amino acids. This membrane EF-Tu accounted for 5-10% of total cell EF-Tu. When spheroplasts were pretreated with trypsin, EF-Tu in the outer membrane disappeared. Incubation of cytosol EF-Tu with the outer membrane did not result in the binding of EF-Tu to the membrane. These results indicate that the appearance of EF-Tu in the outer membrane is not due to artificial binding during membrane preparation. It is suggested that the ribosomal alteration resulted in dislocation of the cytosol protein into the outer membrane.     

Transmembrane orientation of glycoproteins encoded by the v-fms oncogene

The retroviral oncogene v-fms encodes a glycoprotein whose transport to the plasma membrane is required for transformation. Tryptic digestion of microsomes from transformed cells yielded membrane-protected amino-terminal fragments 40 kd smaller than intact molecules. These fragments were glycosylated, and they included v-fms-coded epitopes expressed at the cell surface. Deletion of the predicted membrane-spanning peptide generated polypeptides that were completely sequestered within microsomes. The mutant glycoproteins acquired more asparagine-linked oligosaccharide chains than did wild-type molecules, lacked kinase activity in vitro, were not transported to the cell surface, and had no transforming activity. Thus, the membrane-spanning segment in the middle of the glycoprotein interrupts translocation of nascent chains into the endoplasmic reticulum, ultimately orienting the amino-terminal domain outside the cell and the carboxy-terminal kinase domain in the cytoplasm. These topological features are similar to those of several growth factor receptors, suggesting that v-fms transforms cells through modified receptor-mediated signals.     

Reaction of ozone with glycophorin in solution and in lipid vesicles.

Glycophorin from human red blood cells was exposed to ozone in aqueous solution. Amino acid analysis of glycophorin exposed to a 10-fold molar excess of ozone showed that the only residue affected was methionine. Both methionine residues of the protein were oxidized to methionine sulfoxide. Exposure of the oxidized protein to cyanogen bromide caused no cleavage of the polypeptide chain. Glycophorin was incorporated into unilamellar lipid vesicles made from phosphatidylcholine. The protein containing vesicles were exposed to ozone in a 10-fold molar excess to the glycophorin. Gas chromatography of the methyl esters showed negligible change in the fatty acid composition. Amino acid analysis of the ozone-treated protein showed the oxidation of only one methionine residue per polypeptide chain to methionine sulfoxide. Ghosts of human erythrocytes were exposed to ozone. Cyanogen bromide treatment of the oxidized glycophorin yielded fragments showing that the only methionine residue oxidized by ozone was residue 8. These results indicate that in this membrane model (a) amino acid is more susceptible to ozone than is the lipid, and (b) amino acids external to the membrane are more susceptible than those in the polypeptide chain spanning the membrane.     

Saccharomyces cerevisiae phospholipid:diacylglycerol acyl transferase (PDAT) devoid of its membrane anchor region is a soluble and active enzyme retaining its substrate specificities

A N-terminal deleted version of the Saccharomyces cerevisiae phospholipid:diacylglycerol acyltransferase (ScPDAT), lacking the predicted membrane-spanning region, was fused in frame with alpha-factor secretion signal and expressed in Pichia pastoris under the control of the methanol inducible alcohol oxidase promoter. This resulted in a truncated, soluble and highly active PDAT protein secreted into the culture medium of the recombinant cells. The soluble as well as native membrane bound enzymes was shown to be glycosylated and extensive deglycosylation severely lowered the activity. The production of a soluble and extracellular PDAT allowed us to investigate substrate preferences of the enzyme without interference of endogenous lipids and enzymes. Similar to the membrane bound counterpart, the highest activity was achieved with acyl groups at sn-2 position of phosphatidylethanolamine as acyl donor and 1,2-diacylglycerols as acyl acceptor. The soluble enzyme was also able to catalyze, at a low rate, a number of transacylation reactions between various neutral lipids and between polar lipids and neutral lipids others than diacylglycerols, including acylation of long chain alcohols.     

Peptidoglycans synthesized by a membrane preparation of Micrococcus luteus.

By incubation of cell-free particulate preparations from Micrococcus luteus with nucleotidic precursors uridine 5'-diphosphate-N-acetylglucosamine and uridine 5'-diphosphate-N-acetylmuramic acid-L-Ala-D-iso-Glu-L-Lys-D-Ala-D-Ala, several types of peptidoglycans were obtained: soluble peptidoglycan, insoluble peptidoglycan bound to the membrane and solubilized by trypsin, and peptidoglycan, which remained insoluble after the action of trypsin. The structure of each type of peptidoglycan was studied by action of lytic enzymes and separation of the fragments on Sephadex. Soluble peptidoglycans consist of a mixture of un-cross-linked polymers of various molecular weights. Trypsin-solubilized peptidoglycans are also a mixture of polymers of various sizes. They contain a preponderance of un-cross-linked material and some bridges with dimer peptides. Insoluble peptidoglycans, after the action of trypsin, contain about 50% of un-cross-linked peptide residues; in the other moiety, peptide units are cross-linked by D-Ala leads to L-Lys and D-Ala leads to L-Ala bonds which characterize the natural peptidoglycan. Therefore, the cell-free particulate preparation possesses the whole enzymatic system necessary for synthesis of cross-linked peptidoglycan.     

Phosphorylation in membranes of intact human erythrocytes.

Studies of phosphorylation in membranes of intact human erythrocytes were performed by incubating erythrocytes in inorganic [32P]phosphate. Analysis of membrane proteins by polyacrylamide gel electrophoresis showed a pattern of phosphorylation similar to that observed when ghost membranes were incubated with [gamma-32P]ATP. Membrane lipid phosphorylation was also similar in intact cells and ghosts. The most heavily phosphorylated lipid, polyphosphoinositide, was closely associated with glycophorin A, the major erythrocyte membrane sialoglycoprotein obtained when the sialoglycoprotein fraction was isolated by the lithium diiodosalicylate-phenol partition procedure. Only 1 molecule of glycophorin A out of every 100 was found to be phosphorylated, and the phosphate exchange occurred specifically in the COOH-terminal intracellular portion of glycophorin A. These studies show that the human erythrocyte can be used as a model for membrane phosphorylation in an intact cell system.     

Phosphorylation of glycophorin A in membranes of intact human erythrocytes

The qualitative and quantitative contribution of glycophorin A phosphorylation to the general and specific pattern of membrane protein phosphorylation in intact erythrocytes pre-incubated with 32Pi was examined. Intense 32P-labeled bands at 88,000 and 38,000 Mr were identified as phosphorylated glycophorin A dimer and monomer respectively on the basis of several criteria. Quantitatively, phosphorylated glycophorin A dimer accounted for about 70% of 32P in the band 3 region. This value is at least three times that previously reported. The results of ancillary experiments involving selective extraction of ghosts in acidified chloroform/methanol solvents and electrophoresis in the presence of detergents make it unlikely that the 32P associated with glycophorin A was due to bound polyphosphoinositides.     

Quantitative composition and characterization of the proteins in membrane vesicles released from erythrocytes by dimyristoylphosphatidylcholine. A membrane system without cytoskeleton

Membrane vesicles were prepared by incubation of human erythrocytes with dimyristoylphosphatidylcholine [3] and isolated by isopycnic centrifugation on Dextran density gradients. Protein analyses were carried out with crossed immunoelectrophoresis and dodecylsulfate polyacrylamide gel electrophoresis. The right-side-out-oriented membrane vesicles contained membrane and cytoplasmic proteins of the erythrocyte but lacked cytoskeletal components. Comparison of proteins in vesicles and erythrocyte membranes showed that acetylcholinesterase was enriched two to six times in the vesicles relative to both membrane-spanning proteins, band 3, and glycophorin. Two further, hitherto unidentified, sialic acid-containing membrane antigens were found in the vesicles. Both faced the outside of the membranes and were enriched two to seven times. Ankyrin was not present in the membrane vesicles and spectrin could not be detected by dodecylsulfate polyacrylamide gel electrophoresis. We suggest that the redistribution of proteins in the vesicles reflects differences in their interactions with other membrane components and their relative mobility within the erythrocyte membrane.     

Comparison of the Soluble and Membrane-Bound Forms of the Puromycin-Sensitive Enkephalin-Degrading Aminopeptidases from Rat

Enkephalin degradation in brain has been shown to be catalyzed, in part, by a membrane-bound puromycin-sensitive aminopeptidase. A cytosolic puromycin-sensitive aminopeptidase with similar properties also has been described. The relationship between the soluble and membrane forms of the rat brain enzyme is investigated here. Both of these aminopeptidase forms were purified from rat brain and an antiserum was generated to the soluble enzyme. Each of the aminopeptidases is composed of a single polypeptide of molecular mass 100 kilodaltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography. The antisoluble aminopeptidase antiserum reacts with both enzyme forms on immunoblots and inhibits both with nearly identical inhibition curves. The isoelectric points (pI = 5.0) of both forms were shown to be identical. N-terminal sequencing yielded a common sequence (P-E-K-R-P-F-E-R-L-P-T-E-V-S-P-I-N-Y) for both enzyme forms, and peptide mapping yielded 26 peptides that also appeared identical between the two enzyme forms. Studies on the nature of the association of the membrane enzyme form with the cell membrane suggest that this enzyme form does not represent the soluble form trapped during the enzyme preparation. It is suggested that the membrane form of the puromycin-sensitive aminopeptidase is identical to the soluble enzyme and that it associates with the membrane by interactions with other integral membrane proteins.     

A Ca2+-dependent tryptic cleavage site and a protein kinase A phosphorylation site are present in the Ca2+ regulatory domain of scallop muscle Na+-Ca2+ exchanger

Digestion of scallop muscle membrane fractions with trypsin led to release of soluble polypeptides derived from the large cytoplasmic domain of a Na(+)-Ca(2+) exchanger. In the presence of 1 mm Ca(2+), the major product was a peptide of approximately 37 kDa, with an N terminus corresponding to residue 401 of the NCX1 exchanger. In the presence of 10 mm EGTA, approximately 16- and approximately 19-kDa peptides were the major products. Polyclonal rabbit IgG raised against the 37-kDa peptide also bound to the 16- and 19-kDa soluble tryptic peptides and to a 105-110-kDa polypeptide in the undigested membrane preparation. The 16-kDa fragment corresponded to the N-terminal part of the 37-kDa peptide. The conformation of the precursor polypeptide chain in the region of the C terminus of the 16-kDa tryptic peptide was thus altered by the binding of Ca(2+). Phosphorylation of the parent membranes with the catalytic subunit of protein kinase A and [gamma-(32)P]ATP led to incorporation of (32)P into the 16- and 37-kDa soluble fragments. A site may exist within the Ca(2+) regulatory domain of a scallop muscle Na(+)-Ca(2+) exchanger that mediates direct modulation of secondary Ca(2+) regulation by cAMP.     

Possible identity of a membrane-bound with a soluble cyclic AMP-independent erythrocyte protein kinase that phosphorylates spectrin

A soluble casein kinase isolated and purified to homogeneity from the human erythrocyte cytosol by phosphocellulose and Sephadex G-200 chromatographies is indistinguishable from the membrane-bound casein (spectrin) kinase according to physical and site-specificity criteria. The soluble enzyme shows an Mr of about 30000 by gel filtration and comigrates with the purified membrane spectrin kinase as a single polypeptide of 32000 Da on sodium dodecyl sulfate polyacrylamide gels. The soluble kinase phosphorylates spectrin in situ in spectrin kinase-depleted ghosts and catalyzes the in vitro phosphorylation of partially dephosphorylated spectrin with saturation kinetics identical to those displayed by the membrane spectrin kinase. When component 2 of spectrin that had been phosphorylated with [gamma-32P]ATP by either the soluble or the membrane kinases was subjected to limited proteolysis, the same 21500 Da papain-generated phosphopeptide was found to have been produced by the two enzymes. The same 21500 Da phosphopeptide was identified after papain digestion of spectrin isolated from intact cells that had been incubated with 32Pi. However, this particular peptide was not labeled in spectrin that had been phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. Identical phosphopeptide patterns were obtained by gel filtration and two-dimensional peptide maps of trypsin-cleaved component 2 of spectrin that had been labeled in situ, in intact ghosts or in spectrin kinase-depleted ghosts supplemented with the soluble kinase. These findings indicate a possible identity of the soluble with the membrane-bound casein (spectrin) kinase.     

Biochemical characterization of nuclear pore complex protein gp210 oligomers.

The membrane-spanning glycoprotein gp210 is a major component of the nuclear pore complex. This nucleoporin contains a large cisternal N-terminal domain, a short C-terminal cytoplasmic tail, and a single transmembrane segment. We show here that dimers of native gp210 can be isolated from cell extracts by immunoprecipitation, and from purified rat liver nuclear envelopes by velocity sedimentation and gel filtration. Cross-linking of proteins in isolated membranes prior to solubilization dramatically increases the proportion of dimers. The dimers are SDS-resistant, as previously observed for some integral membrane proteins of cis-Golgi and plasma membrane proteins, including glycophorin A. Larger oligomers of gp210 can also be obtained by gel filtration and denaturing electrophoresis, but unlike the dimers are dissociated by reduction and heating in the presence of SDS. We propose that gp210 is organized into the pore membrane as a large array of gp210 dimers that may constitute a luminal submembranous protein skeleton.