Evidence for covalent attachment of diphytanylglyceryl phosphate to the cell-surface glycoprotein of Halobacterium halobium.

In a previous study, we demonstrated the occurrence of novel proteins modified with a diphytanylglyceryl group in thioether linkage in Halobacterium halobium (Sagami, H., Kikuchi, A., and Ogura, K. (1995) J. Biol. Chem. 270, 14851-14854). In this study, we further investigated protein isoprenoid modification in this halobacterium using several radioactive tracers such as [3H]geranylgeranyl diphosphate. One of the radioactive bands observed on SDS-polyacrylamide gel electrophoresis corresponded to a periodic acid-Schiff stain-positive protein (200 kDa). Radioactive and periodic acid-Schiff stain-positive peptides (28 kDa) were obtained by trypsin digestion of the labeled proteins. The radioactive materials released by acid treatment of the peptides showed a similar mobility to dolichyl (C55) phosphate on a normal-phase thin-layer plate. However, radioactive hydrolysates obtained by acid phosphatase treatment co-migrated not with dolichol (C55-65), but with diphytanylglycerol on both reverse- and normal-phase thin-layer plates. The mass spectrum of the hydrolysate was also coincident with that of diphytanylglycerol. The partial amino acid sequences of the 28-kDa peptides were found in a fragment (amino acids 731-816) obtainable by trypsin cleavage of the known cell-surface glycoprotein of this halobacterium. These results indicate that the cell-surface glycoprotein (200 kDa) is modified with diphytanylglyceryl phosphate.     

cDNA-derived primary structure of the glycoprotein component of canine microsomal signal peptidase complex

Canine microsomal signal peptidase activity has been shown previously to co-migrate as an apparent complex of six polypeptides with molecular masses of 25, 23, 22, 21, 18, and 12 kDa. The 22- and 23-kDa species are differentially glycosylated forms of the same protein, designated SPC 22/23. The amino acid sequence of SPC 22/23 was deduced from cDNA clones. The protein is synthesized without a cleavable amino-terminal signal sequence and contains a single site for N-linked glycosylation. SPC 22/23 appears to be anchored to the rough endoplasmic reticulum membrane by a single hydrophobic segment near its amino terminus, with the remainder of the protein positioned on the lumenal side of the membrane. The amino acid sequence of SPC 22/23 shares homology with tryptic peptides derived from the hen oviduct signal peptidase glycoprotein, one of two possible proteins required for signal peptide processing in the avian system (Baker, R.K., and Lively, M.O. (1987) Biochemistry 26, 8561-8567). Therefore, the complete amino acid sequence of SPC 22/23 presented in this report corresponds to one of two possible proteins required for signal peptide processing in higher eukaryotic cells.     

Trypanosoma brucei brucei and Trypanosoma brucei gambiense: stage specific differences in wheat germ agglutinin binding and in endoglycosidase H sensitivity of glycoprotein oligosaccharides

Gel electrophoresis, lectin affinity blotting, and endoglycosidase H digestion have been used to analyze the glycoprotein profiles of bloodstream and procyclic forms of Trypanosoma brucei brucei and T. b. gambiense. Proteins resolved by polyacrylamide gel electrophoresis were stained with silver nitrate or electrophoretically transferred to nitrocellulose and probed with a horseradish peroxidase conjugate of either concanavalin A or wheat germ agglutinin. Silver staining showed, as expected, that the expression of the variant specific glycoprotein was restricted to the bloodstream forms. Twenty-three concanavalin A binding proteins were resolved in blots of bloodstream forms. Concanavalin A binding molecules corresponding in electrophoretic mobility to 21 of these 23 bloodstream form glycoproteins were detected in blots of procyclic forms. The two concanavalin A binding glycoproteins present only in bloodstream form extracts were variant specific glycoprotein and an 81-kDa protein designated glycoprotein 81b. One concanavalin A binding molecule of 84 kDa, glycoprotein 84p, was detected only in procyclic forms. The 19 major wheat germ agglutinin binding glycoproteins expressed by bloodstream forms were not detected in procyclic forms; only small proteins or protein fragments in procyclic form extracts bound wheat germ agglutinin. Incubating transferred proteins in endoglycosidase H eliminated subsequent binding of concanavalin A to most of the 22 common glycoproteins of bloodstream forms. Three major concanavalin A binding glycoproteins of bloodstream forms, variant specific glycoprotein, glycoprotein 81b, and a 110-kDa molecule (glycoprotein 110b), and other minor glycoproteins carried sugar chains that resisted endoglycosidase H digestion. In contrast, concanavalin A did not bind to any procyclic form glycoproteins, including a 110-kDa concanavalin A binding molecule (glycoprotein 110p) after endoglycosidase H treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis, primary structure and molecular cloning of snowdrop (Galanthus nivalis L.) lectin.

Poly(A)-rich RNA isolated from ripening ovaries of snowdrop (Galanthus nivalis L.) yielded a single 17-kDa lectin polypeptide upon translation in a wheat-germ cell-free system. This lectin was purified by affinity chromatography. Translation of the same RNA in Xenopus leavis oocytes revealed a lectin polypeptide which was about 2 kDa smaller than the in vitro synthesized precursor, suggesting that the oocyte system had removed a 2-kDa signal peptide. A second post-translational processing step was likely to be involved since both the in vivo precursor and the Xenopus translation products were about 2 kDa larger than the mature lectin polypeptide. This hypothesis was confirmed by the structural analysis of the amino acid sequence of the mature protein and the cloned mRNA. Edman degradation and carboxypeptidase Y digestion of the mature protein, and structural analysis of the peptides obtained after chemical cleavage and modification, allowed determination of the complete 105 amino acid sequence of the snowdrop lectin polypeptide. Comparison of this sequence with the deduced amino acid sequence of a lectin cDNA clone revealed that besides the mature lectin polypeptide, the lectin mRNA also encoded a 23 amino acid signal-sequence and a C-terminal extension of 29 amino acids, which confirms the results from in vitro translation experiments.     

Subunit structure of deglycosylated human and swine trachea and Cowper's gland mucin glycoproteins

Summary The oligosaccharide chains in human and swine trachea and Cowper's gland mucin glycoproteins were completely removed in order to examine the subunit structure and properties of the polypeptide chains of these glycoproteins. The carbohydrate, which constitutes more than 70% of these glycoproteins, was removed by two treatments with trifluoromethanesulfonic acid for 3 h at 3° and periodate oxidation by a modified Smith degradation. All of the sialic acid, fucose, galactose, N-acetylglucosamine and N-acetylgalactosamine present in these glycoproteins was removed by these procedures.The deglycosylated polypeptide chains were purified and characterized. The size of the monomeric forms of all three polypeptide chains were very similar. Data obtained by gel filtration, release of amino acids during hydrolysis with carboxypeptidase B and gel electrophoresis in the presence of 0.1% dodecyl sulfate showed that a major fraction from each of the three mucin glycoproteins had a molecular size of about 67 kDa. All of the deglycosylated chains had a tendency to aggregate. Digestion with carboxypeptidases showed that human and swine trachea mucin glycoproteins had identical carboxyl terminal sequences, -Val-Ala-Phe-Tyr-Leu-Lys-Arg-COOH. Cowper's gland mucin glycoprotein had a similar carboxyl terminal sequence, -Val-Ala-Tyr-Leu-Phe-Arg-Arg-COOH. The yield of amino acids after long periods of hydrolysis with carboxypeptidases showed that at least 85% of the polypeptide chains in each of the deglycosylated preparations have these sequences. These results suggested that the polypeptide chains in these deglycosylated mucin glycoprotein preparations were relatively homogeneous.The deglycosylated polypeptide chains as well as the intact mucin glycoproteins had blocked amino terminii. The purified polypeptide chains were digested with trypsin-TCPK, and S. aureus V8 protease and the resulting peptides were isolated by gel electrophoresis in the presence of 0.1% dodecyl sulfate and by HPLC. Two partial amino acid sequences from swine trachea mucin glycoprotein, two partial sequences from human trachea mucin glycoprotein and three partial sequences from Cowper's gland mucin glycoprotein were determined. The partial amino acid sequences of the peptides isolated from swine trachea mucin glycoprotein showed more than 70% sequence homology to a repeating sequence present in porcine submaxillary mucin glycoprotein. Five to eight immunoprecipitable bands with sizes ranging from about 40 kDa to 46 kDa were seen when the polypeptide chains were digested with S. aureus V8 protease. All of the bands had blocked amino terminii and differed by a constant molecular weight of about 1.5 kDa. These data suggest that the polypeptides were formed by cleavage of glutamic acid residues present at regular intervals in the chains of all three mucin glycoproteins. These large immunoreactive peptides were formed by the removal of smaller peptides from the carboxyl terminal end of the deglycosylated mucin glycoprotein chains. Taken collectively, these findings indicate that the polypeptide chains in these mucin glycoproteins are very similar in subunit structure and that there is a high degree of homology between their polypeptide chains.     

Identification and partial purification of a band 3-like protein from rabbit renal brush border membranes

A monoclonal antibody against the membrane domain of human erythrocyte band 3 was tested for its ability to bind to rabbit renal brush border membranes. A single brush border protein with a molecular mass of 43 kDa was recognized by the band 3 antibody. Using DNase I coupled to an agarose-bead support this 43-kDa protein was partially purified by removing actin and a number of actin-bound proteins from the brush border membranes. The partially purified 43 kDa-band was eluted from sodium dodecyl sulfate-polyacrylamide gels and used to make a highly sensitive and specific guinea pig antiserum. This antiserum, but not serum from control guinea pigs, cross-reacts with purified band 3 from human, rabbit, and bovine erythrocytes confirming the immunologic similarity among these proteins. The 43-kDa protein can be stained by the periodic acid-Schiff base method and binds wheat germ agglutinin and concanavalin A, demonstrating that it is a glycoprotein. Furthermore, in the absence of dithiothreitol, the immunoreactive brush border protein migrates with a molecular mass of 86 kDa on an sodium dodecyl sulfate-polyacrylamide gel suggesting that under nonreducing conditions it exists as a dimer. The 43-kDa protein could be solubilized in octyl glucoside and was further purified using gel filtration chromatography. The amino acid composition of the 43-kDa brush border protein was obtained, and its similarity with erythrocyte band 3 is discussed.     

Isolation and characterization of rat lung Pneumocystis carinii gp120

The principal glycoprotein (gp120) of Pneumocystis carinii obtained from infected rat lung was isolated by differential extraction and size-exclusion chromatography. The purified glycoprotein was cleaved with CNBr to two peptides of approximately 27 and 33 kDa. Amino acid sequences were obtained from both peptides. Proteolytic digestion with V8 protease yielded several peptides and sequences were obtained from peptides of 10 and 19 kDa. The cyanogen bromide cleavage results led to the conclusion that gp120 exists as a homodimer.     

Isolation and characterization of calcium binding glycoproteins of cardiac sarcolemmal vesicles

Two major Ca2(+)-binding glycoproteins Mr 120,000 and 100,000 were isolated from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid -solubilized bovine heart sarcolemma membrane. Peroxidase-conjugated concanavalin A and wheat germ agglutinin lectins bind strongly to the isolated 120- and 100-kDa glycoproteins. Treatment with endoglycosidase F resulted in conversion of the 120-kDa glycoprotein to a form migrating at about 97 kDa. Treatment of the 100-kDa band with endoglycosidase F produced form of about 80 kDa. Endoglycosidase H digestion removes only 5% of the mass of both glycoproteins. the carbohydrate structure of both glycoproteins, is therefore, predicted to be at least 75% complex structure and 25% high mannose or hybrid structure. The 120- and 100-kDa glycoproteins are the major Ca2(+)-binding proteins in the sarcolemma membranes. Intact and endoglycosidase-treated glycoproteins bind 45Ca2+ as analyzed by a 45Ca2+ overlay technique. Using polyclonal antibodies, the 120- and 100-kDa glycoproteins were identified in muscle plasma membranes (ventricles, atria, and uterus smooth muscle). They were, however, not present in non-muscle tissues such as pancreas, liver, and kidney. The 120- and 100-kDa glycoproteins appear to be homologous molecules as judged by their similar V8 protease peptide maps, cross-reactivity with polyclonal antibody, and other physicochemical properties.     

Cell-free synthesis, membrane integration, and glycosylation of pro-sucrase-isomaltase

Cell-free translation of total RNA from rabbit intestinal mucosa in a rabbit reticulocyte lysate, after immunoprecipitation with antibodies directed against sucrase-isomaltase, yielded a polypeptide of 200 kDa, which was identified as pro-sucrase-isomaltase. Addition of dog pancreatic microsomal vesicles to the translation system resulted in the appearance of an additional 220-kDa polypeptide. The 220-kDa polypeptide was associated with the membranes in a way that made it inaccessible to proteolysis; this protection was abolished by lytic detergent concentrations, indicating that the polypeptide was segregated into the microsomal vesicle. The 220-kDa polypeptide was glycosylated as evidenced by it being bound to concanavalin A-Sepharose and eluted with alpha-methyl-D-mannopyranoside. The increase in apparent molecular mass (approximately 20 kDa) of the primary translation product upon translocation was due to the addition of carbohydrate; treatment of the 220-kDa polypeptide with endo-beta-N-acetylglucosaminidase H increased its electrophoretic mobility to that of the 200-kDa polypeptide which was obtained in the absence of membranes. Partial N-terminal amino acid sequence of a translation product labeled with [3H]Leu in the absence of membranes revealed that Leu was incorporated into identical positions as in the final (pro)-sucrase-isomaltase, thus indicating the lack of a transient signal peptide.     

Biosynthesis and intracellular processing of carbonic anhydrase in Chlamydomonas reinhardtii

Carbonic anhydrase (CA) of Chlamydomonas reinhardtii is a glycoprotein of 35 kDa which is localized outside the plasma membrane. The activity of CA was increased when the CO2 concentration during photoautotrophic growth was decreased to air level. After decreasing the CO2 concentration from 4% to 0.04%, several polypeptides including CA were induced continuously or transiently. To investigate the biosynthesis and intracellular processing of CA, the cells of wall-less mutant CW-15, which secretes CA into the culture medium, were pulse-labeled with radioactive arginine, chased, and radioactive proteins were immunoprecipitated with anti-CA serum. A 42-kDa polypeptide with isoelectric point (pI) of 7.1-7.3 was first synthesized. Within 5 min the molecular mass of this polypeptide was decreased to 35 kDa and it was then secreted into the culture medium within 30 min. This indicates that the former is the precursor form and the latter the mature form of CA. The primary translation product from poly(A)-rich RNA in a cell-free reticulocyte lysate system from a rabbit was a 38-kDa polypeptide. This was cotranslationally converted into the 42-kDa precursor in vitro in the presence of dog pancreatic microsomal membranes. As the 42-kDa precursor had a high affinity to concanavalin A, it was assumed to have a high-mannose-type oligosaccharide. The mature enzyme had a pI of 6.1-6.2 and was composed of more than two isoforms, which had a complex-type oligosaccharide with low affinity to concanavalin A. Chemical deglycosylation of the mature enzyme by trifluoromethanesulfonic acid indicated that the molecular mass of the polypeptide moiety was 32 kDa and the difference between this and the primary translation product suggests that cleavage of the polypeptide occurs during its biosynthesis.     

Identification of Treponema pallidum subspecies pallidum genes encoding signal peptides and membrane-spanning sequences using a novel alkaline phosphatase expression vector

Treponema pallidum subspecies pallidum is a pathogenic spirochaete for which there are no systems of genetic exchange. In order to provide a system for the identification of T. pallidum surface proteins and potential virulence factors, we have developed a novel expression vector which confers the utility of TnphoA transposition. The relevant features of this plasmid vector, termed pMG, include an inducible tac promoter, a polylinker with multiple cloning sites in three reading frames, and an alkaline phosphatase (AP) gene lacking the signal sequence-encoding region. Library construction with Sau3A-digested T. pallidum genomic DNA resulted in the creation of functional T. pallidum-AP fusion proteins. Analysis of fusion proteins and their corresponding DNA and deduced amino acid sequences demonstrated that they could be grouped into three categories: (i) those with signal peptides containing leader peptidase I cleavage sites, (ii) those with signal peptides containing leader peptidase II cleavage sites, and (iii) those with non-cleavable hydrophobic membrane-spanning sequences. Triton X-114 detergent phase partitioning of individual T. pallidum-AP fusions revealed several clones whose AP activity partitioned preferentially into the hydrophobic detergent phase. Several of these fusion proteins were subsequently shown to be acylated by Escherichia coli following [3H]-palmitate labelling, indicating their lipoproteinaceous nature. DNA and amino acid sequence analysis of one acylated fusion protein, Tp75, confirmed the presence of a hydrophobic N-terminal signal sequence containing a consensus leader peptidase II recognition site. The DNA sequence of Tp75 also indicates that this is a previously unreported T. pallidum lipoprotein. T. pallidum-AP fusion proteins which partitioned into the hydrophobic detergent phase but did not incorporate palmitate were also identified. DNA and amino acid analysis of one such clone, Tp70, showed no cleavable signal but had a significant hydrophobic region of approximately 20 residues, consistent with a membrane-spanning domain. Immunoblot analysis of T. pallidum-AP fusions detected with a monoclonal antibody specific for AP identified several fusion proteins which migrated as doublets separated in apparent electrophoretic mobility by no more than 3 kDa. [35S]-methionine pulse-chase incorporation showed that the doublet AP fusions represented precursor and processed forms of the same protein. DNA and amino acid sequence analysis of clones expressing processed fusion proteins demonstrated hydrophobic N-terminal signal sequences containing consensus leader peptidase I recognition sites.     

Localization of the peptidase activity of human serum butyrylcholinesterase in a approximately 50-kDa fragment obtained by limited alpha-chymotrypsin digestion

Purified human serum butyrylcholinesterase (approximately 90-kDa subunit) is known to exhibit aryl acylamidase and peptidase activity. Limited alpha-chymotrypsin digestion of the purified butyrylcholinesterase gave three major protein fragments of approximately 50 kDa, approximately 21 kDa and approximately 20 kDa. In our earlier studies [Rao and Balasubramanian (1989) Eur. J. Biochem. 179, 639-644] we characterized the approximately 20-kDa fragment and showed that it exhibited both butyrylcholinesterase and aryl acylamidase activities. In the present studies the approximately 50-kDa fragment is characterized. This fragment, after isolation by Sephadex G-75 chromatography from a chymotryptic digest of purified butyrylcholinesterase, exhibited only peptidase activity and was devoid of cholinesterase and aryl acylamidase activities. It could bind to a column of Ricinus communis agglutinin bound to Sepharose, indicating its glycosylated nature and the presence of galactose. The peptidase activity in the approximately 50-kDa fragment could be immuno-precipitated by a polyclonal antibody raised against purified butyrylcholinesterase. SDS-gel electrophoresis of this fragment isolated by R. communis agglutinin-Sepharose and Sephadex G-75 chromatography showed a protein band of approximately 50 kDa by silver staining. Amino-terminal sequence analysis of the approximately 50-kDa fragment gave the sequence of Gly-Pro-Thr-Val-Asp which corresponded to amino acid residues 291-295 in the butyrylcholinesterase sequence [Lockridge et al. (1987) J. Biol. Chem. 262, 549-557]. The combined results suggested that alpha-chymotrypsin digestion of human serum butyrylcholinesterase resulted in the formation of a approximately 20-kDa fragment exhibiting both cholinesterase and aryl acylamidase activities and a approximately 50-kDa fragment exhibiting only peptidase activity.     

Identification and expression of a human cytomegalovirus early glycoprotein.

A human cytomegalovirus early gene which possesses three temporally regulated promoters is located in the large unique component of the viral genome between 0.054 and 0.064 map units (C.-P. Chang, C.L. Malone, and M.F. Stinski, J. Virol. 63:281-290, 1989). This gene contains a major open reading frame (ORF) located 233 bases downstream of the cap site of an early unspliced RNA. The major ORF predicts a polypeptide of 17 kilodaltons (kDa) which contains a glycoproteinlike signal and anchor domains as well as potential N-glycosylation sites. Antisera were prepared against synthetic peptides derived from amino acid sequences within the major ORF. The antisera detected a viral glycoprotein of 48 kDa in infected cells and recognized the in vitro-translated 17-kDa protein early-gene product. The viral glycoprotein, designated gp48, was modified by N-linked glycans and possibly O-linked glycans. The synthesis of gp48 occurred in the absence of viral DNA replication but accumulated to the highest levels at late times after infection. Since gp48 was found in the virion, it is considered an early structural glycoprotein.     

Structure and expression of an inhibitor of fungal polygalacturonases from tomato

A polygalacturonase inhibitor protein (PGIP) was characterized from tomato fruit. Differential glycosylation of a single polypeptide accounted for heterogeneity in concanavalin A binding and in molecular mass. Tomato PGIP had a native molecular mass of 35 to 41 kDa, a native isoelectric point of 9.0, and a chemically deglycosylated molecular mass of 34 kDa, suggesting shared structural similarities with pear fruit PGIP. When purified PGIPs from pear and tomato were compared, tomato PGIP was approximately twenty-fold less effective an inhibitor of polygalacturonase activity isolated from cultures of Botrytis cinerea. Based on partial amino acid sequence, polymerase chain reaction products and genomic clones were isolated and used to demonstrate the presence of PGIP mRNA in both immature and ripening fruit as well as cell suspension cultures. Nucleotide sequence analysis indicates that the gene, uninterrupted by introns, encodes a predicted 36.5 kDa polypeptide containing amino acid sequences determined from the purified protein and sharing 68% and 50% amino acid sequence identity with pear and bean PGIPs, respectively. Analysis of the PGIP sequences also revealed that they belong to a class of proteins which contain leucine-rich tandem repeats. Because these sequence domains have been associated with protein-protein interactions, it is possible that they contribute to the interaction between PGIP and fungal polygalacturonases.     

Active site-directed photoaffinity labeling and partial characterization of oligosaccharyltransferase

Oligosaccharyltransferase, the enzyme that catalyzes the transfer of the oligosaccharide chain of dolichol-P-P-GlcNAc2Man9Glc3 to asparagine residues in -Asn-X-Thr/Ser- sites within polypeptides, has been radiolabeled using a photoactivatable azido tripeptide acceptor, N alpha-[3H]Ac-Asn-Lys(N epsilon-p-azidobenzoyl)-Thr-NH2. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular mass of the oligosaccharyltransferase polypeptide from hen oviduct microsomes is 60 kDa. Radiolabeling of the 60-kDa polypeptide was completely dependent upon photolysis of hen oviduct endoplasmic reticulum preparations in the presence of the azido peptide and Mn2+, which is required for enzymatic activity. Labeling of the enzyme was not inhibited in the presence of a 10-fold excess of the nonacceptor peptides, unacetylated Asn-Lys(N epsilon-p-azidobenzoyl)-Thr-NH2 or unacetylated Asn-Leu-Thr-NH2, whereas it was completely abolished by the presence of a 10-fold excess of the competing acceptor peptide, N alpha-Bz-Asn-Leu-Thr-NH2. Thermal inactivation of oligosaccharyltransferase was achieved by heating endoplasmic reticulum preparations to 60 degrees C. This loss of enzyme activity at 60 degrees C paralleled a comparable decrease in radiolabeling of the 60-kDa polypeptide, whereas temperatures of 50 degrees C and lower had no effect on either process. Oligosaccharyltransferase itself may be an N-linked glycoprotein, because the 60-kDa radiolabeled polypeptide binds to concanavalin A-agarose and is susceptible to digestion by beta-endohexosaminidase H.     

Cloning and expression of male-specific protein (MSP) from the hemolymph of greater wax moth, Galleria mellonella L

Male-specific protein (MSP) is a soluble protein that accumulates in high amounts in the hemolymph and other organs of adult male wax moth. The MSP was purified from adult male wax moth by gel filtration and reversed phase column chromatography, and its amino acid sequence was determined. Because of blocked N-terminus, several internal amino acid sequences of MSP were obtained by the in-gel digestion method using trypsin. RT-PCR was conducted using degenerate primers designed from the internal amino acid sequences. 5'-RACE PCR was used to obtain the complete coding region and 5'-UTR sequence. The full length MSP cDNA sequence encodes a 239 amino acid polypeptide with an 18 amino acid signal peptide. The putative mature MSP has a molecular mass of 24,317 Da and an isoelectric point (pI) of 6.00, but shows a molecular mass of 27 kDa on SDS-PAGE. Sequence alignment showed a significant similarity between MSP and juvenile hormone binding proteins (JHBPs) of several lepidopteran species, including G. mellonella.     

Cytoplasmic orientation and two-domain structure of the multidrug transporter, P-glycoprotein, demonstrated with sequence-specific antibodies

The predicted cytoplasmic orientation and two-domain structure of the multidrug efflux pump P-glycoprotein were demonstrated with sequence-specific antibodies. We synthesized peptides corresponding to amino acid residues, Glu393-Lys408 (anti-P) and Leu1206-Thr1226 (anti-C) in P-glycoprotein from human mdr1 cDNA and used these peptides to produce polyclonal antibodies. From the primary structure of P-glycoprotein, and anti-C antibody is expected to recognize another position, Leu561-Thr581, in the duplicate structure of P-glycoprotein, but anti-P recognizes only one site. These antibodies bind to multidrug-resistant cells (KB-C2) with permeabilized plasma membrane but do not bind to nonpermeabilized KB-C2 cells or parental KB cells, supporting the predicted cytoplasmic orientation of these sequences. With immunoblotting of the membrane fractions from KB-C2 cells, a major 140-kDa polypeptide of the P-glycoprotein was detected with both anti-P and anti-C. Two minor polypeptides with molecular mass of 95 and 55 kDa were also detected. When membrane vesicles were digested mildly with trypsin, the amount of these two polypeptides increased. Anti-P detected only the 95-kDa polypeptide, and anti-C detected both 95- and 55-kDa polypeptides. Achromobacter lyticus protease I (lysyl endopeptidase) and Staphylococcus aureus V8 protease also produced two polypeptides with similar molecular weights. Absorption into lectin-agarose beads and labeling with [3H]glucosamine indicated that the 95-kDa polypeptide was glycosylated but that the 55-kDa polypeptide was not. These two polypeptides as well as P-glycoprotein were photoaffinity-labeled with a calcium channel blocker, [3H]azidopine, but most of the label was found in the 55-kDa polypeptide. The yield of labeled fragments from membrane vesicles photolabeled after digestion with trypsin was similar to that from membrane vesicles digested with trypsin after photolabeling. These data indicate 1) that the 95-kDa polypeptide is the fragment corresponding to the amino-terminal half of P-glycoprotein containing sugar chains; 2) that the 55-kDa polypeptide is the carboxyl-terminal half which was mainly labeled with [3H]azidopine; and 3) that P-glycoprotein has a relatively rigid structure with a small number of protease-sensitive sites and its global structure is not destroyed by tryptic cleavage.     

Amino acid sequence studies on cyanogen bromide peptides of chicken caldesmon which bind to calmodulin

Three major calmodulin-binding cyanogen bromide peptides (fragments A, B, and D) were isolated from chicken gizzard muscle caldesmon and their amino acid sequences were determined. The molecular masses of fragments A, B, and D were estimated to 16, 12, and 9 kDa, respectively, by SDS-urea polyacrylamide gel electrophoresis. Fragment A was composed of 102 amino acid residues and contained homoserine at the C terminus. The amino acid sequence from the 37th residue of fragment A corresponds to the N-terminal sequence of the 15 kDa peptide which was obtained by thrombin digestion [Mornet, D., Audemard, E., & Derancourt, J. (1988) Biochem. Biophys. Res. Commun. 154, 564-571]. Thrombin 15 kDa peptide binds to F-actin but does not bind to calmodulin. Thus the N-terminal 36 residues and the C-terminal part from the 37th residue of fragment A are supposed to bind to calmodulin and F-actin, respectively. The sequences of fragments B and D were identical, but fragment D was composed of 64 amino acid residues and ended with tryptophan, whereas fragment B was of 98 or 99 amino acid residues and ended with proline. Both fragments B and D are supposed to be the C-terminal peptides of chicken caldesmon. Fragment B had heterogeneous sequences at the C-terminal region. These results can explain the reported heterogeneity of chicken caldesmon in charge and molecular mass.