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1.
We have recently demonstrated that the 1CF11 monoclonal antibody bound human milk lactoferrin (hLf) through the recognition of two distinct portions of the molecule, namely the N-glycan-relevant and -irrelevant structural elements. In this present study, we prepared four immunoreactive peptide fractions containing N-linked glycan from tryptic digests of reduced and alkylated hLf by using a concanavalin A lectin column and reverse-phase HPLC. Deglycosylation of these fractions and a competitive binding assay using fucosylated oligosaccharides revealed that the non-reducing terminal fucose residue in N-linked glycan(s) played a significant role in recognizing the N-glycan-relevant element in hLf by 1CF11.  相似文献   

2.
Monoclonal antibody 1CF11 has been suggested to specifically recognize a certain carbohydrate epitope shared by glycoproteins in human external secretions. We examined the effect of cleaving the polypeptide backbone and removing N-linked oligosaccharides on the reactivity with 1CF11 of human milk lactoferrin (hLf) to elucidate the structural features of the 1CF11 epitope. We reveal by treating hLF with trypsin and/or N-glycosidase that both the N-glycan-relevant and N-glycan-irrelevant structural elements were involved in the recognition of hLf by 1CF11.  相似文献   

3.
We have shown that recombinant forms of VP8* domains of the human rotavirus outer capsid spike protein VP4 from human neonatal strains (N155(G10P[11]) and RV3(G3P[6]) and a bovine strain (B223) recognize unique glycans within the repertoire of human milk glycans. The accompanying study by Yu et al.2, describes a human milk glycan shotgun glycan microarray that led to the identification of 32 specific glycans in the human milk tagged glycan library that were recognized by these human rotaviruses. These microarray analyses also provided a variety of metadata about the recognized glycan structures compiled from anti-glycan antibody and lectin binding before and after specific glycosidase digestions, along with compositional information from mass analysis by matrix-assisted laser desorption ionization-mass spectrometry. To deduce glycan sequence and utilize information predicted by analyses of metadata from each glycan, 28 of the glycan targets were retrieved from the tagged glycan library for detailed sequencing using sequential disassembly of glycans by ion-trap mass spectrometry. Our aim is to obtain a deeper structural understanding of these key glycans using an orthogonal approach for structural confirmation in a single ion trap mass spectrometer. This sequential ion disassembly strategy details the complexities of linkage and branching in multiple compositions, several of which contained isomeric mixtures including several novel structures. The application of this approach exploits both library matching with standard materials and de novo approaches. This combination together with the metadata generated from lectin and antibody-binding data before and after glycosidase digestions provide a heretofore-unavailable level of analytical detail to glycan structure analysis. The results of these studies showed that, among the 28 glycan targets analyzed, 27 unique structures were identified, and 23 of the human milk glycans recognized by human rotaviruses represent novel structures not previously described as glycans in human milk. The functional glycomics analysis of human milk glycans provides significant insight into the repertoire of glycans comprising the human milk metaglycome.  相似文献   

4.
A 10-mer random peptide library displayed on filamentous bacteriophage was used to determine the molecular basis of the interaction between the monoclonal anti-colicin A antibody 1C11 and its cognate epitope. Previous studies established that the putative epitope recognized by 1C11 antibody is composed of amino acid residues 19-25 (RGSGPEP) of colicin A. Using the phage display technique it was confirmed that the epitope of 1C11 antibody was indeed restricted to residues 19-25 and the consensus motif RXXXPEP was identified. Shorter consensus sequences (RXXPEP, RXXEP, KXXEP) were also selected. It was also demonstrated that the disulfide bond found in one group of the selected peptides was crucial for 1C11 antibody recognition. It was shown that cyclization of the peptides by disulfide bond formation could result in a structure that mimics the natural epitope of colicin A.  相似文献   

5.
The neural cell adhesion molecule and the voltage-sensitive sodium channel alpha-subunit are the only two molecules in mammals known to be modified by alpha-2,8-linked polysialic acid (polySia). We found a new polySia-containing glycoprotein in human milk and identified it as CD36, a member of the B class of the scavenger receptor superfamily. The polySia-containing glycan chain(s) were removed by alkaline treatment but not by peptide:N-glycanase F digestion, indicating that milk CD36 contained polySia on O-linked glycan chain(s). Polysialylation of CD36 occurs not only in human milk but also in mouse milk. However, CD36 in human platelets is not polysialylated. PolySia CD36 is secreted in milk at any lactation stage and reaches peak level at 1 month after parturition. Thus, it is suggested that polySia of milk CD36 is significant for neonatal development in terms of protection and nutrition.  相似文献   

6.
Angiotensin I-converting enzyme (ACE, CD143) has two homologous domains, each having a functional active site. Fine epitope mapping of 8 mAbs to the C-terminal domain of human ACE was carried out using plate precipitation assays, mAbs' cross-reactivity with ACE from different species, site-directed mutagenesis, and antigen- and cell-based ELISAs. Almost all epitopes contained potential glycosylation sites. Therefore, these mAbs could be used to distinguish different glycoforms of ACE expressed in different tissues or cell lines. mAbs 1B8 and 3F10 were especially sensitive to the composition of the N-glycan attached to Asn 731; mAbs 2H9 and 3F11 detected the glycosylation status of the glycan attached to Asn 685 and perhaps Asn1162; and mAb 1E10 and 4E3 recognized the glycan on Asn 666. The epitope of mAb 1E10 is located at the N-terminal end of the C domain, close to the unique 36 amino acid residues of testicular ACE (tACE). Moreover, it binds preferentially to tACE on the surface of human spermatozoa and thus may find application as an immunocontraceptive drug. mAb 4E3 was the best mAb for quantification of ACE-expressing somatic cells by flow cytometry. In contrast to the other mAbs, binding of mAb 2B11 was not markedly influenced by ACE glycosylation or by the cell culture conditions or cell types, making this mAb a suitable reference antibody. Epitope mapping of these C-domain mAbs, particularly those that compete with N-domain mAbs, enabled us to propose a model of the two-domain somatic ACE that might explain the interdomain cooperativity. Our findings demonstrated that mAbs directed to conformational epitopes on the C-terminal domain of human ACE are very useful for the detection of testicular and somatic ACE, quantification using flow cytometry and ELISA assays, and for the study of different aspects of ACE biology.  相似文献   

7.
Monoclonal antibody 1CF11 has been suggested to specifically recognize a certain carbohydrate epitope shared by glycoproteins in human external secretions. We examined the effect of cleaving the polypeptide backbone and removing N-linked oligosaccharides on the reactivity with 1CF11 of human milk lactoferrin (hLf) to elucidate the structural features of the 1CF11 epitope. We reveal by treating hLF with trypsin and/or N-glycosidase that both the N-glycan-relevant and N-glycan-irrelevant structural elements were involved in the recognition of hLf by 1CF11.  相似文献   

8.
We have recently demonstrated that the 1CF11 monoclonal antibody bound human milk lactoferrin (hLf) through the recognition of two distinct portions of the molecule, namely the N-glycan-relevant and -irrelevant structural elements. In this present study, we prepared four immunoreactive peptide fractions containing N-linked glycan from tryptic digests of reduced and alkylated hLf by using a concanavalin A lectin column and reverse-phase HPLC. Deglycosylation of these fractions and a competitive binding assay using fucosylated oligosaccharides revealed that the non-reducing terminal fucose residue in N-linked glycan(s) played a significant role in recognizing the N-glycan-relevant element in hLf by 1CF11.  相似文献   

9.
Ependymins, a family of extracellular glycoproteins of goldfish and mammalian brain, were shown to contain N-linked complex glycan chains. These glycoproteins reacted with a monoclonal antibody, HNK-1 which recognizes a membrane antigen on a subset of human lymphocytes, myelin-associated glycoprotein glycoprotein epitope reacting with HNK-1 antibody was previously shown to include a terminal 3-sulfoglucuronosyl residue present in certain glycolipids of the nervous tissue (Chou et al., Biochem. Biophys. Res. Commun. 1985, 128, 383-388). In this report, the presence of glucuronic acid in ependymins was demonstrated by gas-liquid chromatography and mass spectrometry. We suggest that a 3-sulfoglucuronosyl residue may be the common epitope on HNK-1-reactive glycoproteins.  相似文献   

10.
Monoclonal antibody TEC-02, raised against mouse embryonal carcinoma cells, has been shown to react with murine preimplantation embryos and with a very limited number of adult mouse tissues. The target epitope, TEC-2, is a carbohydrate carried in mouse embryonal carcinoma cells by large glycoprotein-bound glycan. We report here the expression of TEC-2 epitope on human carcinoma-derived cell lines, HeLa and HS, and the properties of its carbohydrate carriers. Immunolabeling of Nonidet P-40 lysates of HeLa cells separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that TEC-2 antigens are polydispersed glycoconjugates of high molecular weight (mostly above 100,000). TEC-2 antigens detected by the two-site sandwich assay, in which the antigen is immobilized and detected with the same antibody, had a slightly higher molecular weight than those detected by the solid-phase assay. This suggests heterogeneity in the number of TEC-2 epitopes per carrier molecule. When the cells were lysed by Triton X-114 and the detergent and aqueous phases were separated by warming and centrifugation, most of the TEC-2 antigenic activity was found in the aqueous phase. TEC-2 antigens isolated by indirect precipitation from [3H]galactose-labeled HeLa cells were degraded by extensive pronase digestion or mild alkaline treatment to glycopeptides or oligosaccharides of low molecular weight. Thus, TEC-2 epitope in human HeLa cells is carried by carbohydrates of only several monosaccharide units. TEC-02 antibody was also found to bind to Tamm-Horsfall glycoprotein isolated from human urine and its binding was enhanced by desialylation. Combined data indicate that TEC-02 antibody recognizes the GalNAc beta 1----4Gal beta 1----4 structure which may be carried on different types of molecule, according to the site of their synthesis.  相似文献   

11.
We examined a large number of individual human and animal saliva samples for the reactivity with 1CF11, a mouse monoclonal antibody previously produced for the characterization of human milk mucin and apparently recognizing a certain carbohydrate antigenic structure shared by various human glycoproteins in secretions. The results obtained here confirm the unique occurrence of 1CF11 epitope in each and every saliva sample from humans and Old world monkeys as well, though a vast variety was observed among individual saliva samples in the immunological reactivity with 1CF11.  相似文献   

12.
The glycan shield of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) protein serves as a barrier to antibody-mediated neutralization and plays a critical role in transmission and infection. One of the few broadly neutralizing HIV-1 antibodies, 2G12, binds to a carbohydrate epitope consisting of an array of high-mannose glycans exposed on the surface of the gp120 subunit of the Env protein. To produce proteins with exclusively high-mannose carbohydrates, we generated a mutant strain of Saccharomyces cerevisiae by deleting three genes in the N-glycosylation pathway, Och1, Mnn1, and Mnn4. Glycan profiling revealed that N-glycans produced by this mutant were almost exclusively Man(8)GlcNAc(2), and four endogenous glycoproteins that were efficiently recognized by the 2G12 antibody were identified. These yeast proteins, like HIV-1 gp120, contain a large number and high density of N-linked glycans, with glycosidase digestion abrogating 2G12 cross-reactivity. Immunization of rabbits with whole Delta och1 Delta mnn1 Delta mnn4 yeast cells produced sera that recognized a broad range of HIV-1 and simian immunodeficiency virus (SIV) Env glycoproteins, despite no HIV/SIV-related proteins being used in the immunization procedure. Analyses of one of these sera on a glycan array showed strong binding to glycans with terminal Man alpha1,2Man residues, and binding to gp120 was abrogated by glycosidase removal of high-mannose glycans and terminal Man alpha1,2Man residues, similar to 2G12. Since S. cerevisiae is genetically pliable and can be grown easily and inexpensively, it will be possible to produce new immunogens that recapitulate the 2G12 epitope and may make the glycan shield of HIV Env a practical target for vaccine development.  相似文献   

13.
The human immunodeficiency virus type 1 (HIV-1) envelope spike is a heavily glycosylated trimeric structure in which protein surfaces conserved between different HIV-1 isolates are particularly well hidden from antibody recognition. However, even variable regions on the spike tend to be less antigenic and immunogenic than one might have anticipated for external structures. Here we show that the envelope spike of primary viruses has an ability to restrict antibody recognition of variable regions. We show that access to an artificial epitope, introduced at multiple positions across the spike, is frequently limited, even though the epitope has been inserted at surface-exposed regions on the spike. Based on the data, we posit that restricted antibody access may be the result, at least in part, of a rigidification of the epitope sequence in the context of the spike and/or a highly effective flexible arrangement of the glycan shield on primary viruses. Evolution of the HIV envelope structure to incorporate extra polypeptide sequences into nominally accessible regions with limited antibody recognition may contribute to reducing the magnitude of antibody responses during infection and allow the virus to replicate unhindered by antibody pressure for longer periods.  相似文献   

14.
In foregoing work, we identified at least 5 distinct epitopes on human type II collagen (CII), using 8 murine monoclonal antibodies (mAb) against human CII, and suggested that a species-nonspecific epitope on CII recognized by anti-CII mAb termed 1-5 is an arthritogenic epitope. We also found that antibody response against a selected epitope of human CII could be induced by immunization with rabbit anti-idiotypic (Id) antibody against anti-CII mAb. The author developed and characterized monoclonal anti-Id antibodies against 1-5 mAb recognizing a putative arthritogenic epitope. The author also investigated whether the anti-Id mAb could regulate antibody response directed against a selected epitope recognized by 1-5 mAb, and the induction of collagen-induced arthritis in DBA/1J mice. DBA/1J mice intravenously preinjected with anti-Id mAb to 1-5, did not produce anti-CII antibody expressing 1-5 Id upon immunization with human CII. Furthermore, as the development of collagen-induced arthritis (CIA) in DBA/1J mice pretreated with anti-Id mAb to 1-5 was significantly suppressed, anti-Id mAb will be a useful tool for studying the regulation of antibody response to a selected epitope. This study lends support to our hypothesis that the 1-5 epitope is an arthritogenic epitope.  相似文献   

15.
Although human serum albumin is synthesized without carbohydrate, glycosylated variants of the protein can be found. We have determined the structure of the glycan bound to the double-mutant albumin Redhill (-1 Arg, 320 Ala-->Thr). The oligosaccharide was released from the protein using anhydrous hydrazine, and its structure was investigated using neuraminidase and a reagent array analysis method, which is based on the use of specific exoglycosidases. The glycan was shown to be a disialylated biantennary complex type oligosaccharide N-linked to 318 Asn. However, a minor part (11 mol%) of the glycan was without sialic acid. The structure is principally the same as that of glycans bound to two other types of glycosylated albumin variants. Glycosylation can affect, for example, the fatty acid binding properties of albumin. Taking the present information into account, it is apparent that different effects on binding are caused not by different glycan structures but by different locations of attachment, with the possible addition of local conformational changes in the protein molecule.  相似文献   

16.
We examined a large number of individual human and animal saliva samples for the reactivity with ICF11, a mouse monoclonal antibody previously produced for the characterization of human milk mucin and apparently recognizing a certain carbohydrate antigenic structure shared by various human glycoproteins in secretions. The results obtained here confirm the unique occurrence of ICF11 epitope in each and every saliva sample from humans and Old world monkeys as well, though a vast variety was observed among individual saliva samples in the immunological reactivity with ICF11.  相似文献   

17.
A monoclonal antibody (MAb) 5H11/B1 that reacts with a repeating epitope on an excretory-secretory (E + S) antigen of adult worms of Schistosoma mansoni was used in the detection of circulating antigen (CA) in sera from S. mansoni-infected mice using an antigen-capture sandwich ELISA. Trichloroacetic acid (TCA) pretreatment of sera from mice infected for 8 or 16 weeks precipitated immune complexes and/or dissociated CA and allowed its detection. Sera obtained 8 weeks after infection contained high levels of CA. Upon treatment with praziquantel (100 mg/kg body wt), this level was significantly less within 1 week. A strong correlation was found between the worm count determined by perfusion and the level of antigenemia detected by the 5H11/B1 assay in light and heavy infection (r = 0.80). Based on the results of both TCA pretreatment and sodium periodate treatment, the 5H11/B1 sandwich ELISA assay detects a repeating carbohydrate epitope on an E + S antigen. This system appears to be a sensitive assay for the detection of schistosomal antigenemia in murine schistosomiasis. Studies on the detection of antigenemia in human schistosomiasis using this assay are in progress.  相似文献   

18.
In order to count Clostridium tyrobutyricum spores in milk after membrane filtration, murine 21E7-B12 monoclonal antibody was produced. Elution of the monoclonal antibody from this antigen, the flagellar filament protein, by carbohydrate ligands was used to study the epitope structure. A competitive elution of an anti-dextran monoclonal antibody by carbohydrate ligands served as a control in order to validate the immunological tool applied to flagellin epitope study. The carbohydrate moiety of flagellin contained D -glucose and N-acetyl-glucosamine in a molar ratio of 11:1 as determined by gas-liquid chromatography and 2 low-abundancy unidentified compounds. In ELISA, D -glucose and N-acetyl-glucosamine did not dissociate the antibody-flagellin complex contrary to maltose, maltotriose, maltotetraose and maltopentaose. The efficiency of elution increased from the dimer to the pentamer and became nil for maltohexaose and maltoheptaose. The fact that the hexamer and heptamer could not react with the 21E7-B12 monoclonal antibody could be explained by a drastic conformational change. The overall stretched maltopentaose switch to a helical-shaped maltoheptaose which could not fit the 21E7-B12 monoclonal antibody antigen-combining site. Thus, flagellin epitope may contain α(1→4) linked glucose residues plus either N-actyl-glucosamine or an unidentified compound that maintain it in an extended shape.  相似文献   

19.
While isolating free secretory component (FSC) by monoclonal antibody affinity chromatography, we demonstrated FSC-IgG complexes in human milk. We hypothesized that IgG antibody to secretory component (SC) might be transported into the milk from the serum. We therefore examined sera from 10 normal adults and 10 infants for IgG capable of binding to FSC in an enzyme-linked immunosorbent assay. Eight of 10 normal adult sera and nine of 10 infant sera demonstrated IgG binding to FSC with titers ranging from 1:54 to 1:4096. Quantitation of the IgG bound to FSC was hampered in adult sera by the binding of IgM and polymeric IgA to the FSC. Quantitation in five infant sera ranged from 0.5 to 6.4 micrograms/ml. A pepsin digest of an IgG fraction of serum demonstrated binding of the F(ab')2 fragments to the FSC. The specificity of the antibodies in human serum was evaluated by examining the binding to secretory IgA (sIgA) and FSC isolated from pooled human milk and polymeric IgA isolated from the ascitic fluid of a patient with an IgA myeloma. Eight of the 10 adults had antibody specific for FSC. Three of the eight, all female, also had antibody specific for sIgA. Two of the eight had antibody either to FSC and sIgA or to FSC plus an antibody that could bind to an epitope shared by sIgA and FSC. Competition experiments with monoclonal antibodies to human secretory component and sIgA were used to confirm and further define these specificities. The results of this study indicate that antibody to SC is common in normal adult and infant sera. The majority of antibodies seem to be directed against epitopes present on FSC but not on sIgA, which suggests sensitization to circulating or membrane-bound SC. The significance of these antibodies in normal human sera remains to be elucidated.  相似文献   

20.
Glycosylation determines essential biological functions of epithelial mucins in health and disease. We report on the influence of glycosylation of the immunodominant DTR motif of MUC1 on its antigenicity. Sets of novel glycopeptides were synthesized that enabled us to examine sole and combined effects of peptide length (number of repeats) and O-glycosylation with GalNAc at the DTR motif on the binding patterns of 22 monoclonal antibodies recognizing this motif. In case of unglycosylated peptides almost all antibodies bound better to multiple MUC1 tandem repeats. Glycosylation at the DTR led to enhanced binding in 11 cases, whereas 10 antibodies were not influenced in binding, and one was inhibited. In nine of the former cases both length and DTR glycosylation were additive in their influence on antibody binding, suggesting that both effects are different. Improved binding to the glycosylated DTR motif was exclusively found with antibodies generated against tumor-derived MUC1. Based on these data a tumor-specific MUC1 epitope is defined comprising the ...PDTRP... sequence in a particular conformation essentially determined by O-glycosylation at its threonine with either GalNAcalpha1 or a related short glycan. The results can find application in the field of MUC1-based immunotherapy.  相似文献   

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