Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.52 (PNGase F)
 1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The major cat allergen, Fel d I, was purified to homogeneity from cat dander extract by sequential mAb affinity chromatography and HPLC size exclusion. The purity and allergenic activity of the preparation was demonstrated by different techniques such as HPLC, RAST inhibition, skin prick tests and CIE/CRIE. Fel d I showed a mol. wt of about 35,000 by HPLC gel filtration and of 18,000 by SDS-PAGE, confirming that it is a non-covalently linked dimer. However, SDS-PAGE analysis under reducing conditions as well as labelling experiments with 14C-iodoacetamide of 2-ME-reduced Fel d I showed that each mol. wt 18,000 monomer is comprised of two covalently S-S bound polypeptides with apparent mol. wt. of 4000 (alpha-chain) and 14,000 (beta-chain). Reduction and alkylation of Fel d I obliterated most of its allergenic activity, as determined by RAST inhibition and immunoblotting, suggesting that most of the IgE-binding sites are conformational. On the other hand, treatment of Fel d I by N-glycanase under reducing and non-reducing conditions indicated the presence of N-linked oligosaccharides in the beta-chain. Carbohydrate analysis data of the whole Fel d I molecule showed the presence of a relatively high carbohydrate content (approximately 20%). RAST inhibition experiments of native and deglycosilated allergen suggest that most IgE epitopes are located in the protein moiety of the molecule. However, the deglycosilated allergen showed a 2-4 fold reduction in its inhibition capacity of RAST as compared to the native allergen, suggesting that carbohydrates could have some role in keeping the active conformation of those epitopes. The N-terminal amino acid sequence of the beta-chain (20 residues) and most of the alpha-chain (40 residues) were determined. Both chain sequences showed no homology with other known protein sequences.
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PMID:Studies on the biochemical structure of the major cat allergen Felis domesticus I. 171 68

As tools to study structural relationships of cobra venom factor (CVF) and human complement component C3, murine monoclonal antibodies to CVF were produced. In this paper we describe two of these monoclonal anti-CVF antibodies designated GV1.8 and GV1.10, both of which bind to carbohydrate epitopes. On immunoblotting, antibody GV1.8 binds to both the alpha- and beta-chains of CVF, whereas antibody GV1.10 binds only to the alpha-chain of CVF. After enzymatic deglycosylation of CVF with N-glycanase (peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase), both antibodies lose their ability to bind to the deglycosylated protein. Additionally, the free oligosaccharide chains of CVF are able to inhibit the binding of antibodies GV1.8 and GV1.10 to CVF on enzyme-linked immunosorbent assay, further demonstrating their carbohydrate specificity. Both monoclonal antibodies to CVF cross-react with human C3. Antibody GV1.8 binds to both chains of human C3 indicating that the shared antigenic epitope present on the two glycosylated chains of CVF is also present on the two chains of human C3. Antibody GV1.10 cross-reacts only with the beta-chain of human C3 which is the homologous chain to the alpha-chain of CVF. After enzymatic deglycosylation of human C3 by N-glycanase, both antibodies lose their ability to bind to the deglycosylated protein consistent with the carbohydrate nature of the recognized epitopes. These results indicate that CVF and human C3 share carbohydrate epitopes on their homologous and nonhomologous chains.
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PMID:Cobra venom factor and human C3 share carbohydrate antigenic determinants. 244 Sep 48

The mechanisms by which a stimulatory monoclonal antibody (mAb), called mAb F11, induces granular secretion and aggregation in human platelets have been characterized. Fab fragments of mAb F11, as well as an mAb directed against the platelet Fc gamma RII receptor (mAb IV.3) were found to inhibit mAb F11-induced platelet secretion and aggregation, indicating that the mAb F11 IgG molecule interacts with the Fc gamma RII receptor through its Fc domain and with its own antigen through its Fab domain. The mAb F11 recognized two platelet proteins of 32 and 35 kDa on the platelet membrane surface, as identified by Western blot analysis. We purified both proteins from human platelet membranes using DEAE-Sepharose chromatography followed by mAb F11 affinity chromatography. When added to platelet-rich plasma, the purified proteins dose-dependently inhibited mAb F11-induced platelet aggregation. The purified protein preparation also competitively inhibited the binding of 125I-labelled mAb F11 to intact platelets. The N-terminal 26 amino acid sequences of both the 32 and 35 kDa proteins were identical and contained a single unblocked serine in the N-terminal position. When digested with N-glycanase, the 32 and 35 kDa proteins were converted into a single approximately 29 kDa protein, indicating that these two proteins are derived from the same core protein but differ in their degree of glycosylation. Internal amino acid sequence analysis of the F11 antigen provided information concerning 68 amino acids and suggested two consensus phosphorylation sites for protein kinase C (PKC). The phosphorylation by PKC of the isolated F11 antigen was observed following stimulation by phorbol 12-myristate 13-acetate. Databank analysis of the N-terminal and internal amino acid sequences of the F11 antigen indicated that the N-terminal sequence exhibited the highest degree of similarity to the variable region of the alpha-chain of human T-cell receptors (TCR). In contrast, the F11 internal sequences did not exhibit any similarity to the TCR. Our results demonstrate that the F11 antigen is a novel platelet membrane surface glycoprotein which becomes cross-linked with the Fc gamma RII receptor when platelets are activated by the stimulatory mAb F11. These mechanisms may be relevant to the production of immune thrombocytopenia by platelet-activating antibodies.
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PMID:Mechanisms of platelet activation by a stimulatory antibody: cross-linking of a novel platelet receptor for monoclonal antibody F11 with the Fc gamma RII receptor. 764 39

The structures of the N-linked oligosaccharides on recombinant human hepatocyte growth factor (rh-HGF) expressed by Chinese hamster ovary (CHO) cells were studied by two-dimensional sugar mapping. The oligosaccharides released from the glycopeptides by peptide: N-glycosidase F (PNGase F) treatment were tagged with 2-aminopyridine at the reducing ends. The alpha-chain was linked by biantennary, triantennary, and tetraantennary oligosaccharides, but the dominant oligosaccharides linking the beta-chain were biantennary (> 85%). There was no significant difference in oligosaccharide structures between the two glycosylation sites on each chain, that is, Asn263 and Asn371 on the alpha-chain, and Asn535 and Asn622 on the beta-chain. The linkage of sialic acid to the non-reducing terminal galactose was identified as NeuAc alpha(2-3) by 1H-NMR spectrometry. The structures of the N-linked oligosaccharides from rat HGF were also studied. Triantennary oligosaccharides were obtained from the alpha-chain and a biantennary oligosaccharide was obtained from the beta-chain. This result indicates that the alpha-chain is also linked by higher branched oligosaccharides than the beta-chain in rat HGF.
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PMID:Structural study of the N-linked oligosaccharides of hepatocyte growth factor by two-dimensional sugar mapping. 840 80

This study was undertaken to investigate the nature and microheterogeneity of the carbohydrate moiety of the Fc epsilon receptors of RBL-CA10 and RBL-CA10.7 cells. Treatment using the glycosylation processing inhibitors, castanospermine (CN), 1-deoxymannojirimycin (DMJ), and swainsonine (SW) resulted in a decrease of the relative molecular mass (M(r)) of both the alpha-chain of the high affinity receptor for IgE, Fc epsilon RI(alpha), and the low affinity receptor for IgE, Fc epsilon RL. Exposure to DMJ had the greatest effect on the M(r), while CN seemed to lead to a decreased cell surface expression of Fc epsilon RI. Both receptors are largely resistant to endoglycosidase H as their M(r) decreased only by approximately 2 kDa. These results suggest that both receptors are composed primarily of complex oligosaccharides with a single high mannose, N-glycosylated site. Both Fc epsilon receptors become endoglycosidase H sensitive if first exposed to DMJ which indicates that the carbohydrate composition is indeed altered by this processing inhibitor presumably by blocking the formation of complex structures. When the Fc epsilon receptors were reduced and hydrolyzed by N-glycanase, the M(r) values for Fc epsilon RI(alpha) and Fc epsilon RL decreased to approximately 28 and 34-38 kDa respectively. In the case of Fc epsilon RI(alpha), this implies the presence of only a small amount of O-linked oligosaccharides.
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PMID:The N-linked oligosaccharides of the Fc epsilon receptors of rat basophilic leukemia cells. 843 10