Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Two protein antigens were isolated from excretory-secretory products of Trichinella spiralis by biochemical methods and characterized with respect to their chemical and immunological properties. One antigen, of apparent Mr 43,000, is an abundant secreted protein of infective L1 larvae, while the other, of 45-50 kDa, is present in smaller amounts. Yields, extinction coefficients, isoelectric points, amino acid compositions, and partial N-terminal amino acid sequences for each are reported. Partial amino acid sequences of peptides derived from the 43-kDa protein by cyanogen bromide cleavage have been determined. Treating a reduced-pyridylethylated derivative of the 43-kDa protein with glycopeptidase F (N-glycanase) resulted in formation of a transient product of 37 kDa followed by a stable polypeptide of 32 kDa (by SDS-PAGE), suggesting the presence of two N-linked carbohydrate groups. A similar result was obtained with the 45-50-kDa protein, which gave a transient doublet of 38 and 40 kDa and a final, stable product of 33 kDa, with a minor component of 35 kDa. Two glycosylation sites of the 43-kDa protein and one site of the 45-50-kDa protein can be identified in the amino acid sequences. Polyclonal antibodies prepared against the two proteins cross-reacted extensively, but failed to react with the doubly deglycosylated polypeptides in Western blots. The dominant epitopes present in the reduced-pyridylethylated polypeptides are, therefore, N-linked carbohydrate, although the presence of peptide epitopes in the native proteins cannot be excluded.
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PMID:Partial characterization of two antigens secreted by L1 larvae of Trichinella spiralis. 239 16

A plasmid pAc373GM-CSF was constructed and co-transfected into Spodoptera frugiperda (Sf9) cells with wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. The recombinant virus vAc373GM-CSF was identified and purified by several rounds of plaque hybridization. By assaying the culture medium, we demonstrated recombinant virus infected Sf9 cells expressing hGM-CSF. Recombinant hGM-CSFs with apparent molecular masses of 14.5, 15.5 and 16.5 kDa were detected by the Western blot method. All 3 forms have biological activity of hGM-CSF. Following N-glycanase treatment, a single band of 14.5-15.5 kDa appeared in SDS-PAGE. Western blot analysis of expression in Sf9 cell treated with tunicamycin revealed only the presence of the 14.5 kDa species. Thus, the signal sequence of recombinant hGM-CSF could be recognized and cleaved by infected insect cell and the resultant molecule secreted into the media.
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PMID:Expression of human granulocyte-macrophage colony-stimulating factor gene in insect cells by a baculovirus vector. 240 25

A variety of homobifunctional crosslinking agents have been used to gain insight into the nature of the murine interleukin 3 (mIL-3) receptor. When [125I]mIL-3 was cross-linked to receptor sites on the surfaces of intact B6SUtA1 cells with disuccinimidyl suberate (DSS), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed the existence of two radiolabeled species with molecular weights of 140 (p140) and 70 (p70) kd (after subtraction of [125I]mIL-3). The relative intensities of the two bands did not change when the [125I]mIL-3 concentration was varied, confirming Scatchard results which suggested only one affinity class. However, when [125I]mIL-3 was crosslinked to intact cells and then incubated at 37 degrees C, the intensity of p140 decreased relative to p70, suggesting a conversion of p140 to p70. This conversion could be inhibited by sodium azide, methylamine, and bacitracin and could also be prevented by first boiling for 1 min in 2% SDS and 5% 2-mercaptoethanol. The putative protease that carried out this apparent conversion appeared to be associated both with plasma membranes prepared from these cells and also with solubilized receptors. Moreover, when p140, crosslinked with both dithiobis succinimidylpropionate and glutaraldehyde, was purified and reelectrophoresed under reducing conditions, p70 could be generated. N-glycanase digestion of p140 and p70 revealed a similar level of N-linked carbohydrate, which upon closer study appeared to consist of two chains, a 3-kd and an 8-kd moiety. Consistent with this data, we propose that the receptor is a 140-kd glycoprotein that is cleaved to a 70-kd surface protein upon mIL-3 binding and chemical crosslinking.
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PMID:Characterization of the interleukin 3 receptor. 240 77

Mutation of the 3' splice sites bordering exon 2 of the H-2Dd and H-2Kd genes generated alternatively spliced transcripts when the constructs were transfected into L cells (J. Immunol. 143:1018). The H-2Dd transcripts contained an additional 84 nucleotides derived from the first intervening sequence, whereas 60 extra bases were included in the H-2Kd mRNA. Proteins derived from these transcripts were recognized by mAb. Moreover, both Ag served as recognition elements for CTL, and the mutant H-2Kd molecule functioned as a restricting element for an Ag peptide. As a result of alternative splicing, the mutant proteins should have additional residues at their NH2 termini to increase their lengths by 28 (Dd) or 20 (Kd) amino acids. Immunoprecipitation and analysis on SDS-PAGE demonstrated that the mutant H-2Kd molecule was indeed larger than the normal H-2Kd protein, but the mutant and wild-type H-2Dd Ag were the same size. In addition, treatment of H-2Dd mutant and normal Ag with N-glycanase produced molecules of equal size, demonstrating that the mutant protein was completely glycosylated. Limited amino acid sequencing of this Ag indicated that it was normal H-2Dd. Therefore, before its transfer to the cell surface, post-translational modifications remove the additional NH2-terminal residues of the mutant Dd but not Kd protein.
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PMID:Expression of mutant H-2d proteins encoded by class I genes which alternatively process the 5' end of their transcripts. 247 8

Human Fc gamma RIII (CD16), a low-affinity receptor expressed on several different cell types, has a polymorphism on polymorphonuclear cells (Fc gamma RIIIPMN) identified by the NA1 and NA2 markers. Inasmuch as this polymorphism has functional consequences, an understanding of the structural biology of Fc gamma RIII may provide important insight into receptor function. We analyzed Fc gamma RIIIPMN by SDS-PAGE and found that receptor from individuals allotyped for either NA1 or NA2 contained only one protein after removal of N-linked glycosylations (19 and 21 kDa respectively) whereas receptor from NA1/2 individuals contained both bands. Because some reports indicate that digestion of Fc gamma RIII on NK cells (Fc gamma RIIINK) with N-glycanase also results in two bands on SDS-PAGE, we investigated Fc gamma RIIINK to explore the possibility of a corresponding allelic polymorphism in this receptor. Contrary to expectation, Fc gamma RIIINK from all donors irrespective of their NA allotype contained two bands (20 and 24 kDa) on SDS-PAGE after deglycosylation. In addition, those distinct epitopes on the extracellular domain of Fc gamma RIIIPMN found with mAb B73.1 and CLB gran 11 in association with the NA allotypic differences are expressed (or not expressed) on Fc gamma RIIINK independent of donor NA allotype. Fc gamma RIIIPMN and Fc gamma RIIINK differ at the protein level as they have different m.w. (glycosylated and deglycosylated), different epitopes in the extracellular domain (not attributable to tissue-specific glycosylation), and differential expression of the NA allelic protein polymorphism. Although the membrane anchor of Fc gamma RIIIPMN is a phosphatidylinositol-specific phospholipase C sensitive glycosyl-phosphatidylinositol linkage, Fc gamma RIIINK is insensitive to phosphatidylinositol-specific phospholipase C. However, a form of Fc gamma RIIINK is released from NK cells upon incubation at 37 degrees C. Thus, the basis for the two bands in Fc gamma RIIINK after N-linked deglycosylation is neither coexpression of two molecular isoforms with different membrane anchors nor an identifiable allelic polymorphism in m.w. restricted to Fc gamma RIIINK (p less than 10(-6)). The differences between the two receptors indicate that, independent of cell anchor type, PMN and mononuclear cells must have different molecular isoforms. The allelic variants, different isoforms, alternative anchor mechanisms and release processes provide for an extensive genetic and regulatory diversity in Fc gamma RIII function.
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PMID:Human Fc gamma RIII (CD16). Isoforms with distinct allelic expression, extracellular domains, and membrane linkages on polymorphonuclear and natural killer cells. 247 7

Human chromosomal DNA encoding single-chain urokinase-type Plasminogen Activator (scu-PA, or pro-urokinase) was inserted in an expression plasmid and transfected in human A431, mouse LB6 and CHO cells. LB6 cells were also transfected with a Bovine Papilloma Virus derivative containing the scu-PA gene. Human scu-PA was purified from cell supernatants of recombinant clones and characterized for structure and function. All recombinant scu-PAs are undistinguishable from human urine-derived scu-PA for peptide backbone, but possess a higher sugar content, as revealed by SDS-PAGE analysis after digestion with glycopeptidase F. This difference is partly due to an increased sialic acid content, as shown by analysis of neuraminidase-treated scu-PAs. No difference was found, however, among recombinant and natural scu-PAs in the kinetics of conversion into two-chain active forms (tcu-PAs) by human plasmin, and in the KM and kcat values of tcu-PA activity on the chromogenic substrate S-2444 and on human plasminogen. Also, recombinant and non-recombinant tcu-PAs displayed similar dose-response curves for binding to the endothelial inhibitor PAI-1. In conclusion, the glycosylation pattern of u-PA does not affect its interaction with the plasma proteins directly involved in its fibrinolytic function.
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PMID:The differential glycosylation of human pro-urokinase from various recombinant mammalian cell lines does not affect activity and binding to PAI-1. 251 81

The major component immunoprecipitated from human red cell membranes by murine monoclonal antibodies (BS46 and BS56) against the LW blood group antigens is a 42,000 mol. wt glycoprotein. Upon digestion by an N-glycanase the LW component migrated as a 25,000 mol. wt component on SDS gels, whereas treatment by an O-glycanase led only to a small size reduction (2000). These data suggest that the LW glycoprotein might carry approximately eight to nine N-linked sugar chains and only a few (one or two) O-linked oligosaccharide chains. A minor component of 31,000 mol. wt was also identified in the LW immunoprecipitate. Preliminary analyses by two-dimensional peptide mapping indicate that the 31,000 mol. wt polypeptide is identical to authentic Rh proteins, therefore raising the possibility that the Rh and LW antigens are associated in the membrane as a functional complex called Rh cluster. Since the N-deglycosylated form of the LW and RhD proteins have different sizes (25,000 vs 31,000-32,000 respectively) and since their externally 125I-labelled domains have different two-dimensional peptide maps, it is concluded that LW is probably not a simple glycosylated form of the Rh proteins.
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PMID:Properties of the blood group LW glycoprotein and preliminary comparison with Rh proteins. 251 51

We characterized Fc receptor III (FcR III) on human neutrophils and found it to be heavily glycosylated and polymorphic. In some individuals, FcR III that had been digested with N-glycanase appeared after SDS-PAGE under reducing conditions as two bands with apparent molecular masses of 33 and 29 kD. In other individuals, N-glycanase-treated FcR III appeared as a single band with an Mr of either 33 or 29 kD. After SDS-PAGE of N-glycanase-treated FcR III under nonreducing conditions, the apparent Mr of each structural type was decreased, suggesting the presence of intramolecular disulfide bonds. Digestion of the 33-kD band and the 29-kD band with Staphylococcus aureus V8 protease yielded similar, but not identical, peptide maps. Thus, at least two polymorphic forms of FcR III are expressed on human neutrophils. The structural polymorphism of neutrophil FcR III correlated with previously described antigenic polymorphisms detected by monoclonal antibody Gran 11 and by alloantisera which recognize epitopes of the biallelic, neutrophil antigen (NA) system. Individuals whose neutrophils expressed the two-band structural type of FcR III were NA1NA2 heterozygotes. Individuals whose neutrophils expressed the single 33-kD band structural type were NA2NA2 homozygotes, and individuals whose neutrophils expressed the single 29-kD band structural type were NA1NA1 homozygotes. These findings indicate that antigenic and structural polymorphisms of human neutrophil FcR III are related and can be accounted for by differences at the level of primary protein structure.
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PMID:Characterization of polymorphic forms of Fc receptor III on human neutrophils. 252 15

gp120 and CD4 are two glycoproteins that are considered to interact together to allow the binding of HIV to CD4+ cells. We have utilized enzymatic digestion by endoglycosidases in order to analyze N-linked carbohydrate chains of these proteins and their possible role in the interaction of gp120 or gp160 with CD4. SDS denaturation was not necessary to obtain optimal deglycosylation of either molecule, but deglycosylation of CD4, nonetheless, depended on the presence of 1% Triton X-100. Endo H and Endo F that cleave high mannose type and biantennary glycans diminish the molecular mass of the glycoproteins from 120 or 160 Kd to 90 or 130 Kd, respectively; but these enzymes had no action on CD4 glycans. Endo F N-glycanase mixture, which acts on all glycan species, including triantennary chains, led to complete deglycosylation of gp120/160 and of CD4. Therefore, probably half of the glycan moieties of gp120/160 are composed of high mannose and biantennary chains, the other half being triantennary species. The carbohydrate structures of CD4 seems to be triantennary chains. To analyze the binding of gp120/160 to CD4, we used a molecular assay in which an mAb (110-4) coupled to Sepharose CL4B allowed the attachment of soluble gp120/160 to the beads; 125I-sCD4 was then added to measure the binding of CD4 to different amounts of gp120/160. Binding to gp160 was not modified when using completely deglycosylated 125I-sCD4, while deglycosylation of gp120 or of gp160 resulted in the decrease of the binding to native CD4 by two- and fivefold, respectively. Native and deglycosylated gp120/160 bound to CD4+ cells with comparable affinities. In addition, deglycosylated gp120 displaced 125I-gp160 binding to CD4+ cells and inhibited fusion of fresh Molt-T4 cells with CEM HIV1- or HIV2-infected cells to the same extent. Taken together, these results indicate that carbohydrates of CD4 and of gp120/160 do not play a significant role in the in vitro interaction between these two molecules.
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PMID:Role of N-linked glycans in the interaction between the envelope glycoprotein of human immunodeficiency virus and its CD4 cellular receptor. Structural enzymatic analysis. 253 47

The activities of a cationic (C.PRX) and an anionic peroxidase isolated from peanut (Arachis hypogaea)-cell suspension culture were drastically reduced when they were deglycosylated with glycopeptidase F or oxidized by 10 mM-periodate. In contrast with the controls, the deglycosylated or the oxidized peroxidases were much more susceptible to proteolytic degradation. In radiolabelling experiments with [35S]methionine, the non-glycosylated C.PRX was synthesized in the tunicamycin-treated cultures and secreted into the medium. Examination of the C.PRX polypeptides by SDS/polyacrylamide-gel electrophoresis followed by fluorography showed that the non-glycosylated form had an Mr of approx. 31,000, which is about 78% of that of the glycosylated form. Our results suggest that carbohydrates may not be essential for peroxidase secretion, but that stabilization of the peroxidase molecules and acquisition by these isoenzymes of a catalytically active conformation is linked directly or indirectly to glycosylation.
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PMID:Role of carbohydrate moieties in peanut (Arachis hypogaea) peroxidases. 260 91


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