共查询到20条相似文献,搜索用时 15 毫秒
1.
U Karsten C Diotel G Klich H Paulsen S Goletz S Müller FG Hanisch 《Canadian Metallurgical Quarterly》1998,58(12):2541-2549
The epithelial mucin MUC1 is an important tumor marker of breast cancer and other carcinomas. Its immunodominant DTR motif, which is the principal target for immunotherapeutic approaches, has been assumed until recently not to be glycosylated in both normal and tumor MUC1 and to acquire its immunogenic conformation by virtue of a certain number of tandem repeats. We present evidence that the antigenicity of the single repeat toward a considerable number of antibodies to the DTR motif is greatly enhanced if it is glycosylated within this motif, and only in this position. Twenty-eight monoclonal anti-MUC1 antibodies with DTR specificity were tested for binding to synthetic 21-mer (AHG21) or 20-mer (HGV20) tandem repeat peptides O-glycosylated with galactose beta1-3N-acetylgalactosamine alpha or N-acetylgalactosamine alpha at defined Thr or Ser positions. Binding was measured in ELISA experiments using the glycopeptides as plate-immobilized antigens or as inhibitors in solution. At least 12 antibodies revealed significantly enhanced binding to the peptides glycosylated at the DTR motif (Thr-10) as compared to positional isomers glycosylated at Thr-5, Ser-6, Ser-16, or Thr-17 and to the nonglycosylated peptides. Six antibodies (VU-3-C6, A76-A/C7, Ma552, VU-11-D1, VU-12-E1, and VU-11-E2) that were unreactive with the monomeric repeat peptide did bind to the DTR-glycosylated peptide. Several lines of evidence suggest that glycosylation with N-acetylgalactosamine is sufficient for the observed enhancement effect. Our results are of special interest in conjunction with the recent observation that the DTR motif of lactation-associated MUC1 is O-glycosylated in vivo (Müller et al., J. Biol. Chem., 272: 24780-24793, 1997). They may have consequences for the design of efficient tumor vaccines. 相似文献
2.
Other laboratories have established that oligosaccharyl transferase (OST) from Saccharomyces cerevisiae can be purified as a protein complex containing eight different subunits. To identify the OST subunit that recognizes the peptide sites that can be glycosylated, we developed photoaffinity probes containing a photoreactive benzophenone derivative, p-benzoylphenylalanine (Bpa), as part of an 125I-labeled peptide that could be expected to be glycosylated. We found that Asn-Bpa-Thr peptides served as substrates for OST and that photoactivation of these probes in the presence of microsomes abolished the OST activity. Photoactivation of 125I-labeled Asn-Bpa-Thr in the presence of microsomes resulted in specific covalent labeling of a protein doublet of molecular mass 62 and 64 kDa. By carrying out the photoactivation of the probe using microsomes containing epitope-tagged Ost1p, we demonstrated that the 125I-labeled protein was Ost1p. Radiolabeling of this protein was dependent on irradiation at 350 nm. No labeling was detected using a probe containing Ala instead of Thr as the third amino acid residue. We conclude that Ost1p is the subunit of the OST complex that recognizes the peptide sites in the nascent chains that are destined to be glycosylated. 相似文献
3.
CJ O''Boyle J MacFie K Dave PS Sagar P Poon CJ Mitchell 《Canadian Metallurgical Quarterly》1998,14(4):358-362
The potential of electrospray mass spectrometry (ESMS) for the sequencing of glycopeptides was evaluated using quadrupole time-of-flight (QTOF) technology in the MS/MS mode. The location of O-glycosylation sites was possible in the positive ion (+) mode by detection of prominent y- and b-fragment ions from the underivatized TAP25-2 [T1APPAHGVT9S10APDT14RPAPGS20T21APPA], an overlapping sequence of MUC1 tandem repeats which had been glycosylated in vitro by two GalNAc residues in the positions T9 and T21. The high mass resolution and accuracy of QTOF-(+)ESMS allowed reliable structural assignments. The reduced complexity of the fragment spectra and the higher signal-to-noise ratio render QTOF-(+)ESMS an alternative mass spectrometric approach to the identification of O-glycosylation sites when compared with sequencing by post-source decay matrix-assisted laser desorption/ionization MS. Diagnostic ions from the N-terminus in the b-series offered direct evidence, which was supported by indirect evidence from the C-terminus ions of the y-series. The higher glycosylated GalNAc2-substituted fragments were mainly observed as multiply ionized species. 相似文献
4.
We have investigated the potential of structural elucidation of O-linked glycopeptides by post-source decay matrix-assisted laser desorption ionization mass spectrometry (PSD-MALDI-MS). In order to establish detailed fragmentation patterns and to dissect fragment ions with and without carbohydrate content, the same O-linked MUC1-derived glycopeptides with acetylated and non-acetylated sugars were analysed and compared. Furthermore, we were interested to examine possible differences in the fragmentation between glycopeptides with acetylated and non-acetylated sugars. The obtained PSD-MALDI-MS spectra showed a rather complete set of fragmentation data which allows to localize the glycan on the peptide, in parallel with sequencing a short glycan and the backbone peptide. Fragmentations of the sugars were dominated by inter-ring cleavages at the glycosidic bond. Intra-ring cleavage did also occur from the non-reducing end, but to a much lower extent. The fragmentation of the peptide backbone was not changed either by acetylated or non-acetylated sugars. Glycosylated peptide fragments occurred as fully glycosylated fragment ions, partially deglycosylated ions and completely deglycosylated ions, and was not influenced by the acetylation of sugars. However, differences occurred in the quality and quantity of fragment ions from the non-reducing end of the glycan part when comparing acetylated with non-acetylated glycopeptides. 相似文献
5.
A secretion leader derived from a domain of the extracellular Barrier protease of the yeast Saccharomyces cerevisiae has been expressed in wild-type and in mnn1, mnn9, and mnn1 mnn9 glycosylation mutant strains of S. cerevisiae. Structural comparison of the extracellular leader by mass spectrometry, peptide mapping, and elementary analysis proved that all strains produced a heterogeneous, heavily glycosylated polypeptide of 161 amino acids with both N- and O-glycosylation and phosphorylation. All three potential Asn N-linked sites were glycosylated to some extent with the expected structures. Neither the different growth media used nor the glycosylation mutations had significant effect on O-glycosylation with respect to both site selectivity and size of the carbohydrate structures. All 33 Ser and 21 Thr residues in the polypeptide were glycosylated at least partially, with an average of more than 2 mannoses/site. Although the mnn1 mutation blocks addition of alpha 1,3-linked mannose, the bar secretion domain expressed in the mnn1 and mnn1 mnn9 transformants unexpectedly contained some O-linked structures with at least 4 mannoses/chain. These O-linked structures were as large as when the leader was expressed in the mnn9 and wild-type strains. The bar secretion domain also had a previously undocumented phosphorylated O-linked structure. 相似文献
6.
Tryptic digests of ovine submaxillary apomucin were fractionated by gel filtration and ion exchange chromatography to give 14 peptide fractions. Three purified tryptic peptides, representing 106 of the 650 residues in apomucin, were submitted to automated sequence analysis. The NH2-terminal 50 of the 74 residues in one peptide and the entire sequence of the other two hexadecapeptides were established. These studies suggest that purified ovine submaxillary, mucin is chemically homogeneous, containing a unique primary structure without substantial repeating sequences in its polypeptide chain. The sequences adjacent to 28 known O-glycosidically substituted seryl and threonyl residues were compared. No homologies were apparent around the glycosylated seryl and threonyl residues which might define the specificity of the UDP-N-acetylgalactosaminyl:mucin polypeptide transferase that incorporates N-acetylgalactosamine into O-glycosidic linkage in glycoproteins. However, there appears to be a minimum size requirement for glycosylation, because the transferase catalyzes glycosylation of tryptic peptides efficiently, while chymotryptic and thermolytic peptides were much poorer substrates for the transferase. 相似文献
7.
OBJECTIVE: To refine the algorithms governing peptide presentation by HLA-B*2705 by analyzing: (i) the specificity of the human transporter associated with antigen processing (TAP) for HLA-B27 binding peptides; and (ii) the peptide binding affinity to HLA-B*2705. METHODS: TAP-translocation was measured with a labeled reporter peptide containing an N-linked glycosylation acceptor site in Streptolysin O-permeabilized cells for a panel of HLA-B27 binding peptides. Peptide binding affinity was determined by peptide-induced stabilization of empty HLA-B*2705 expressed by the TAP-deficient cell line T2-B27. RESULTS: Human TAP preferentially translocated analogues with residues leucine, isoleucine, methionine and arginine as the carboxy-terminal amino acids, whereas analogues with aspartic acid and serine were translocated poorly. The binding affinity to HLA-B*2705 of the poorly translocated aspartic acid and serine analogues was about 100-fold less compared to the parent HLA-B27 binding peptide. CONCLUSIONS: Human TAP shows considerable specificity for the C-terminus of potential HLA-B27 ligands. Nonamer peptides with aspartic acid and serine at the C-terminus are poorly translocated by the TAP and have low binding affinity for HLA-B*2705, and are therefore unlikely to become presented by HLA-B*2705. 相似文献
8.
P Dokurno PA Bates HA Band LM Stewart JM Lally JM Burchell J Taylor-Papadimitriou D Snary MJ Sternberg PS Freemont 《Canadian Metallurgical Quarterly》1998,284(3):713-728
The anti-breast tumour antibody SM3 has a high selectivity in reacting specifically with carcinoma-associated mucin. SM3 recognises the core repeating motif (Pro-Asp-Thr-Arg-Pro) of aberrantly glycosylated epithelial mucin MUC1, and has potential as a therapeutic and diagnostic tool. Here we report the crystal structure of the Fab fragment of SM3 in complex with a 13-residue MUC1 peptide antigen (Thr1P-Ser2P-Ala3P-Pro4P-Asp5P-Thr6P -Arg7P-Pro8P-Ala9P-Pro10P-Gly11P- Ser12P-Thr13P). The SM3-MUC1 peptide structure was solved by molecular replacement, and the current model is refined at 1.95 A resolution with an R-factor of 21.3% and R-free 28.3%. The MUC1 peptide is bound both by non-polar interactions and hydrogen bonds in an elongated groove in the antibody-combining site through interactions with Complimentarity Determining Regions (CDRs), three of the light chain (L1, L2, L3) and two of the heavy chain (H1 and H3). The conformation of the peptide is mainly extended with no discernable standard secondary structure. There is a single non-proline cis-peptide bond in H3 (Val95H-Gly96H-Gln97H-Phe98H-Ala101H-Ty r102H) between Gly96H and Gln97H, which appears to play a role in SM3-peptide antigen interactions, and represents the first such example within an antibody hypervariable loop. The SM3-MUC1 peptide structure has implications for rational therapeutic and diagnostic antibody engineering. 相似文献
9.
Drosocin is a cationic 19 amino acid peptide secreted by Drosophila in response to septic injury. The sequence (GKPRPYSPRPTSHPRPIRV) contains six Pro and four Arg residues which are incorporated into three repeated triplet sequences Pro-Arg-Pro. The peptide is glycosylated at Thr11 and has potent antimicrobial activity. This activity is markedly reduced on deglycosylation, but a structural basis for this has not been previously established. In the current study, the solution conformations of drosocin and its non-glycosylated derivative were determined by NMR spectroscopy and structure calculations. The NMR and structure studies showed that the peptides have significant populations of essentially random coil conformations in aqueous solution. Addition of 50% trifluoroethanol causes the development of small populations of folded conformations, mainly in the form of turns. In particular, turn elements occur near residues 4-7, 10-13, 17, and 18. No substantial difference was detected in the predominantly random coil conformation of the glycosylated and non-glycosylated forms, but there are subtle differences in the small populations of folded conformers. In particular, the turn at residues 10-13 tends toward a more extended structure on glycosylation, while there is some tightening of the downstream turn at residues 17 and 18. There are a significant number of nuclear Overhauser enhancement contacts between the sugar moiety and the peptide near the glycosylation site, consistent with a close association between them. Despite this close association, the pKa of H13, which is proximate to the glycosylation site, was found to be unaffected by glycosylation. 相似文献
10.
V Apostolopoulos V Karanikas JS Haurum IF McKenzie 《Canadian Metallurgical Quarterly》1997,159(11):5211-5218
HLA-A*0201/Kb transgenic mice were immunized with oxidized mannan-mucin 1 (MUC1) as a fusion protein (containing five repeats of the 20-amino-acid MUC1 VNTR (variable number of tandem repeats) that generated highly active CD8+ CTLs to MUC1 peptides. In a direct CTL assay, the MUC1 peptides could be presented specifically by both the transgenic murine HLA-A*0201/Kb and human HLA-A*0201 molecules. The 9-mer MUC1 peptide sequences (APDTRPA and STAPPAHGV) were presented by HLA-A*0201, although they did not contain L at P2 and L/V at P9, the preferred motifs; as a consequence, the binding was of relatively low affinity when compared with a high affinity-binding HIV peptide (ILKEPVHGV). In addition, when mice were immunized separately with the HLA-A*0201-binding peptides (STAPPAHGV or APDTRPAP-containing peptides-keyhole limpet hemocyanin-mannan), direct lysis of MCF-7 (HLA-A*0201+, MUC1+) also occurred. The findings are of interest for tumor immunotherapy, particularly as the CTLs generated to low affinity-binding peptides were highly active and could specifically lyse an HLA-A*0201+ human breast cancer cell line without further in vitro stimulation. The findings demonstrate that the range of peptides that can generate CTLs is broader than formerly considered. 相似文献
11.
EP Bennett H Hassan U Mandel E Mirgorodskaya P Roepstorff J Burchell J Taylor-Papadimitriou MA Hollingsworth G Merkx AG van Kessel H Eiberg R Steffensen H Clausen 《Canadian Metallurgical Quarterly》1998,273(46):30472-30481
A fourth human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T4, was cloned and expressed. The genomic organization of GalNAc-T4 is distinct from GalNAc-T1, -T2, and -T3, which contain multiple coding exons, in that the coding region is contained in a single exon. GalNAc-T4 was placed at human chromosome 12q21.3-q22 by in situ hybridization and linkage analysis. GalNAc-T4 expressed in Sf9 cells or in a stably transfected Chinese hamster ovary cell line exhibited a unique acceptor substrate specificity. GalNAc-T4 transferred GalNAc to two sites in the MUC1 tandem repeat sequence (Ser in GVTSA and Thr in PDTR) using a 24-mer glycopeptide with GalNAc residues attached at sites utilized by GalNAc-T1, -T2, and -T3 (TAPPAHGVTSAPDTRPAPGSTAPPA, GalNAc attachment sites underlined). Furthermore, GalNAc-T4 showed the best kinetic properties with an O-glycosylation site in the P-selectin glycoprotein ligand-1 molecule. Northern analysis of human organs revealed a wide expression pattern. Immunohistology with a monoclonal antibody showed the expected Golgi-like localization in salivary glands. A single base polymorphism, G1516A (Val to Ile), was identified (allele frequency 34%). The function of GalNAc-T4 complements other GalNAc-transferases in O-glycosylation of MUC1 showing that glycosylation of MUC1 is a highly ordered process and changes in the repertoire or topology of GalNAc-transferases will result in altered pattern of O-glycan attachments. 相似文献
12.
M Selby A Erickson C Dong S Cooper P Parham M Houghton CM Walker 《Canadian Metallurgical Quarterly》1999,162(2):669-676
We investigated whether hepatitis C virus envelope glycoprotein E1 is transported from the endoplasmic reticulum (ER) to the cytoplasm of infected cells for class I MHC processing. Target cells expressing E1 were killed by CTL lines from a hepatitis C virus-infected chimpanzee, and synthetic peptides were used to define an epitope (amino acids 233-GNASRCWVA-241) presented by the Patr-B*1601 class I MHC molecule. An unusually high concentration (>100 nM) of this nonameric peptide was required for target cell lysis, but this could be reduced at least 1000-fold by replacing the asparagine at amino acid position 234 (Asn234) with aspartic acid (Asp), the anticipated anchor residue for NH2-terminal peptide binding to Patr-B*1601. Conspicuously, position 234 is part of an N-glycosylation motif (Asn-Xaa-Ser/Thr), suggesting that the Asn234 to Asp substitution might occur naturally within the cell due to deglycosylation/deamidation of this amino acid by the cytosolic enzyme peptide N-glycanase. In support of this model, we demonstrate that presentation of the epitope depended on 1) cotranslational synthesis of E1 in the ER, 2) glycosylation of the E1 molecule, and 3) a functional TAP transporter to shuttle peptide from the cytosolic to ER compartment. These results indicate for the first time that during infection of the host, viral envelope glycoproteins originating in the ER are processed in the cytoplasm for class I MHC presentation. That a posttranslational change in amino acid sequence from Asn to Asp alters the repertoire of peptides presented to CD8+ CTL has implications for the design of antiviral vaccines. 相似文献
13.
Some monoclonal antibodies (MoAbs) directed against blood group M-related epitope of glycophorin A (GPA) were found to agglutinate rare variant erythrocytes carrying GPA of Mg type. In contradistinction to normal GPA-M or -N, the N-terminal portion of GPA-Mg is not glycosylated. Therefore, the multipin peptide synthesis was used for testing the specificity of the cross-reacting MoAbs. Among several anti-M and anti-N MoAbs tested, only three anti-M (E3, E6, 425/2B) agglutinated Mg erythrocytes and showed binding to the synthetic octapeptides corresponding to N-terminal sequences of GPA-M (SSTTGVAM), GPA-N (LSTTEVAM), and GPA-Mg (LSTNEVAM). Testing multiple peptide analogs (window and replacement analysis) showed that these MoAbs were specific for peptidic epitope in which Met8 and Val6 were the most essential amino acid residues. The amino acid replacements Ser<-->Leu1 or Gly<-->Glu5 (M v N) and Thr4<-->Asn4 (M and N v Mg) had no or negligible effect on the reaction of synthetic peptides with the MoAbs. However, when Ser2, Thr3, and Thr4 carry O-linked sialooligosaccharides (normal GPA-M or -N), the MoAbs recognize Gly5- and sialic acid-dependent blood group M-related epitope. An interesting finding concerning anti-M/Mg MoAbs described here is the fact that glycosylation of amino acid residues adjacent to the most important part of peptidic epitope not only differentially modulates the proper exposure of peptidic epitope, but also alters the requirement for some amino acid residues present within the epitope. Pathologic conditions, including hematologic disorders, are often accompanied by alterations in protein glycosylation, resulting not only from differences in the structure of antigen polypeptide chain, but also from changes in specificity or expression of enzymes involved in glycosylation. Our present findings draw attention to possibility of the bidirectional modulation of protein antigenicity by glycosylation and may be helpful in interpretation of some results obtained with MoAb used for diagnostic or other purposes. 相似文献
14.
Cells were treated with two proteolytic inhibitors, N-acetyl-leucyl-leucyl-norleucinal and lactacystin, the latter reported to be a specific inhibitor for the proteasome. Both inhibitors retarded the maturation of endo-H-resistant forms of murine and human class I molecules from their endo-H-sensitive precursors in cell lines with functional TAP proteins. HLA-A2 maturation readily occurs in TAP-deficient T2 cells, and it has been shown that the peptides associated with A2 are derived from the leader segment of proteins in the secretory pathway. This maturation is inhibited by N-acetyl-leucyl-leucyl-norleucinal but not lactacystin, indicating that the proteasome is not required for the generation of HLA-A2 binding peptides in these cells. The murine class Ib molecule Qa-1b presents a leader peptide derived from D-end class I molecules to alloreactive CTL. Since this presentation is dependent on the expression of TAP proteins, we determined if this requirement reflects a need for the proteasome to process this peptide. We found that lactacystin did not inhibit the maturation of endo-H-resistant forms of Qa-1b that are dependent on this leader peptide for its maturation, nor did it inhibit the expression of this peptide-Qa-1b complex in a functional assay. Thus, unlike conventional cytosolic peptides, leader peptides (regardless of whether they are dependent on TAP for their presentation) do not require the proteasome for processing. 相似文献
15.
M Mehta LM White T Knapp RA Kandel JS Wunder RS Bell 《Canadian Metallurgical Quarterly》1998,27(8):427-433
Presentation of antigenic peptides by major histocompatibility complex (MHC) class I molecules depends on translocation of cytosolic peptides into the endoplasmic reticulum (ER) by transporters associated with antigen processing (TAP). Peptide transport by TAP is thought to include at least two steps: initial binding of peptide to TAP, and its subsequent translocation requiring ATP hydrolysis. These events can be monitored in peptide binding and transport assays. Previous studies have shown that the efficiency of peptide transport by human, mouse and rat transporters varies according to the C-terminals of peptide substrates in an allele and species-specific manner. However, it has not been clear during which step of peptide interaction with TAP selection occurs. We used an assay monitoring the peptide binding step to study the binding affinity of a library of 199 peptides for human TAP and the two major allelic rat TAP complexes. We observed a dominant influence of the C-terminus on peptide binding affinity for all transporters, and highly restrictive selection of peptides with aliphatic and aromatic C-terminals by rat TAP1/TAP2u complexes. The selectivity of peptide binding to rat TAP complexes is in full accordance with published data on selective peptide transport and on control of antigen presentation by rat TAP. These results strongly suggest that (i) peptide selection by TAP occurs exclusively in the initial binding step; (ii) all factors involved in peptide selection by TAP are present in insect cells. 相似文献
16.
JL Mellquist L Kasturi SL Spitalnik SH Shakin-Eshleman 《Canadian Metallurgical Quarterly》1998,37(19):6833-6837
Many eukaryotic proteins are modified by Asn-linked (N-linked) glycosylation. The number and position of oligosaccharides added to a protein by the enzyme oligosaccharyltransferase can influence its expression and function. N-Linked glycosylation usually occurs at Asn residues in Asn-X-Ser/Thr sequons where X not equal Pro. However, many Asn-X-Ser/Thr sequons are not glycosylated or are glycosylated inefficiently. Inefficient glycosylation at one or more Asn-X-Ser/Thr sequons in a protein results in the production of heterogeneous glycoprotein products. These glycoforms may differ from one another in their level of expression, stability, antigenicity, or function. The signals which control the efficiency of N-linked glycosylation at individual Asn residues have not been fully defined. In this report, we use a site-directed mutagenesis approach to investigate the influence of the amino acid at the position following a sequon (the Y position, Asn-X-Ser/Thr-Y). Variants of rabies virus glycoprotein containing a single Asn-X-Ser/Thr sequon at Asn37 were generated. Variants were designed with each of the twenty common amino acids at the Y position, with either Ser or Thr at the hydroxy (Ser/Thr) position. The core glycosylation efficiency of each variant was quantified using a cell-free translation/glycosylation system. These studies reveal that the amino acid at the Y position is an important determinant of core glycosylation efficiency. 相似文献
17.
A radioactive peptide has been isolated from cyanogen bromide digests of sodium boro[3H]-hydride-reduced collagen from rabbit bone, tendon and skin. It was identified as a crosslinked peptide linking the short C-terminal cyanogen bromide peptide alpha1-CB6B (17 amino acid residues) to alpha1-CB5 (37 residues) from the helical part of the chain of an adjacent molecule. Both peptides could be separated after cleaving the crosslink with periodate. Thus the crosslinked peptide alpha1-CB5 X alpha1-CB6B originates from an intermolecular crosslink between quarter-staggered molecules within collagen fibrils previously assigned as 'head-to-tail' link. The chemical nature of the reduced crosslinking component was identified and was shown to differ between peptides derived from different tissues: alpha1-CB6B X alpha1 CB5 from bone contains hydroxylysinohydroxynorleucine [o5Lys(o5omegaNle)] whereas the skin peptide contains hydroxylysinonorleucine [o5Lys(omegaNle)]. The peptide derived from tendon contains both components. The relation of o5Lys(omegaNle) to o5Lys(o5omegaNle) in the peptides corresponds to that of the original tissue. On the other hand, histidino-hydroxymerodesmosine which is a major reduced cross-linking component in skin and tendon, is completely absent in the isolated peptides. The crosslinking component in the skin peptide is completely glycosylated, mainly by glucosylgalactosyl residues and to a smaller extent by galactosyl residues. o5Lys(o5omegaNle) from the bone peptide is only partly glycosylated, containing equal amounts of the disaccharide and monosaccharide. Only slight glycosylation was found in the tendon peptide. 相似文献
18.
Relationship between peptide selectivities of human transporters associated with antigen processing and HLA class I molecules 总被引:1,自引:0,他引:1
S Daniel V Brusic S Caillat-Zucman N Petrovsky L Harrison D Riganelli F Sinigaglia F Gallazzi J Hammer PM van Endert 《Canadian Metallurgical Quarterly》1998,161(2):617-624
Efficiency of presentation of a peptide epitope by a MHC class I molecule depends on two parameters: its binding to the MHC molecule and its generation by intracellular Ag processing. In contrast to the former parameter, the mechanisms underlying peptide selection in Ag processing are poorly understood. Peptide translocation by the TAP transporter is required for presentation of most epitopes and may modulate peptide supply to MHC class I molecules. To study the role of human TAP for peptide presentation by individual HLA class I molecules, we generated artificial neural networks capable of predicting the affinity of TAP for random sequence 9-mer peptides. Using neural network-based predictions of TAP affinity, we found that peptides eluted from three different HLA class I molecules had higher TAP affinities than control peptides with equal binding affinities for the same HLA class I molecules, suggesting that human TAP may contribute to epitope selection. In simulated TAP binding experiments with 408 HLA class I binding peptides, HLA class I molecules differed significantly with respect to TAP affinities of their ligands. As a result, some class I molecules, especially HLA-B27, may be particularly efficient in presentation of cytosolic peptides with low concentrations, while most class I molecules may predominantly present abundant cytosolic peptides. 相似文献
19.
The human calcium receptor (hCaR) is a G-protein-coupled receptor containing 11 potential N-linked glycosylation sites in the large extracellular domain. The number of potential N-linked glycosylation sites actually modified, and the effect on cell surface expression and signal transduction of blocking glycosylation at these sites, was examined by site-directed mutagenesis. Asparagine residues of the consensus sequences (Asn-Xaa-Ser/Thr) for N-linked glycosylation were mutated to glutamine individually and in various combinations to disrupt the potential N-linked glycosylation sites in the context of the full-length receptor. The cDNA constructs were transiently transfected into HEK-293 cells lacking endogeneous hCaR, and expressed receptors were analyzed by mobility differences on immunoblots, glycosidase digestion, intact cell enzyme-linked immunoassay, and extracellular calcium-stimulated phosphoinositide hydrolysis assay. Immunoblot analyses and glycosidase digestion studies of the wild type versus mutant receptors demonstrate that, of the 11 potential sites for N-linked glycosylation, eight sites (Asn-90, -130, -261, -287, -446, -468, -488, and -541) are glycosylated; the three remaining sites (Asn-386, -400, and -594) may not be efficiently glycosylated in the native receptor. Sequential mutagenesis of multiple N-linked glycosylation sites and analyses by immunoblotting, immunofluorescence, biotinylation of cell surface proteins, and intact cell enzyme-linked immunoassay indicated that disruption of as few as three glycosylation sites impairs proper processing and expression of the receptor at the cell surface. Disruption of five glycosylation sites reduced cell surface expression by 50-90% depending on which five sites were disrupted. Phosphoinositide hydrolysis assay results for various glycosylation-defective mutant receptors in general correlated well with the level of cell surface expression. Our results demonstrate that among 11 potential N-linked glycosylation sites on the hCaR, eight sites are actually utilized; glycosylation of at least three sites is critical for cell surface expression of the receptor, but glycosylation does not appear to be critical for signal transduction. 相似文献
20.
Y Boulis C Richet K Haupt S Hennebicq B Soudan D Tetaert P Degand MA Vijayalakshmi 《Canadian Metallurgical Quarterly》1998,805(1-2):285-293
A method for the analysis of O-glycosylation of peptides has been developed, combining capillary electrophoretic (CE) separation and electrospray ionization mass spectrometry. Synthetic peptides with apomucin 'tandem repeat' sequences which present potential O-glycosylation sites on threonine and serine residues were used as model system. In vitro O-glycosylated peptide samples were obtained by incubation of the peptides with human gastric microsomal homogenates containing N-acetylgalactosamine transferase activity in the presence of uridyl diphosphate N-acetylgalactosamine (UDP-GalNAc). CE was carried out in the presence of the linear polymer poly(vinyl alcohol) in the electrophoresis solvent, resulting in a greatly improved separation of the up to five different glycoforms of peptides with lengths of 8, 16 or 23 amino acids, and the unglycosylated peptides. After separation and peak collection, the number of modifications with N-acetyl galactosamine (GalNAc) could be determined by electrospray ionization mass spectrometry. The glycosylation pattern was shown to depend on the amino acid sequence of the peptides. 相似文献