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)

Recombinant murine interleukin-4 (muIL-4) expressed in COS-7 monkey kidney cells was purified to homogeneity by sequential CM-Sepharose, Sephadex G-100 chromatography and mono-S FPLC to a specific activity of 6.10(7) units per mg of protein based on an in vitro HT-2 cell proliferation assay. Two electrophoretic variants, designated a and b, which migrated on SDS-PAGE as a closely spaced doublet with Mr 19,000, were present in the final product. Gas phase sequencing of the purified protein revealed the presence of an N-terminus corresponding to the mature protein predicted from the cDNA sequence and sequencing of a cyanogen bromide digest confirmed 75 of the 120 predicted amino acids. Elution behavior on gel filtration corresponded to that of a monomer of Mr 19,000. Since there are three potential sites of N-glycosylation predicted by the cDNA sequence, the contribution of glycosylation to the observed heterogeneity was examined by treatment with endoglycosidases. Variant b was digested by either endo-beta-N-acetylglucosaminidase H (endo H) or endo-beta-N-acetylglucosaminidase F (endo F) to protein of Mr 15,000 on SDS-PAGE but was unaffected by treatment with endo-beta-N-acetylglucosaminidase D (endo D), thus indicating the presence of high mannose type of N-glycan. In contrast, variant a was resistant to endo H, F and D. Complete conversion of a mixture of variants a and b to a single protein of Mr 15,000 on SDS-PAGE was obtained only after treatment with N-glycanase. Both variants were resistant to neuraminidase and O-glycanase treatment. These data show that the microheterogeneity observed in purified muIL-4 preparations is due to differences in the nature of the N-linked oligosaccharides. The availability of purified recombinant muIL-4 and a methodology for both total and selective deglycosylation provides a basis for the initiation of structure-function studies of this novel T-cell lymphokine.
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PMID:Multiple forms of recombinant murine interleukin-4 expressed in COS-7 monkey kidney cells. 278 92

Human DCs (dendritic cells) express surface CD83 upon activation. Comparing the surface induction of CD83 with the upregulation of CD40, CD80 and CD86 during LPS (lipopolysaccharide)-induced DC maturation showed that CD83 induction occurred more rapidly. Despite the lack of CD83 on immature DCs, it was detected in these cells by Western blotting and flow cytometry. Indirect immunofluorescence revealed CD83 inside immature DCs in perinuclear regions. CD83 was absent on monocytes and macrophages, but it was detected inside these cells and found to be rapidly surface-expressed upon LPS-induced activation. Whereas CD83 expression on activated DCs was sustainable, its expression on monocytes and macrophages was transient. Optimal interleukin-4 co-stimulation during DC generation from monocytes was found to be essential for stable CD83 surface expression. CD83 was detected as 37 and 50 kDa forms in transfected 293T cells. Macrophages and immature DCs expressed the 37 kDa form, whereas mature DCs predominantly expressed the 50 kDa form. In monocytes, CD83 was detected as a 22 kDa detergent-insoluble form. The rapid CD83 surface induction on DCs and macrophages was blocked by brefeldin A, but not by cycloheximide, showing that fresh CD83 synthesis was not essential. Tunicamycin inhibited the expression of the 50 and 37 kDa CD83 forms, and also blocked CD83 surface expression on DCs and macrophages. PNGase F (peptide N-glycosidase F) digestion reduced the 37 and 50 kDa CD83 forms to 28 kDa. In summary, monocytes, macrophages and immature DCs contain preformed intracellular CD83, and its rapid surface expression upon activation is post-translationally regulated in a process involving glycosylation.
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PMID:CD83 is preformed inside monocytes, macrophages and dendritic cells, but it is only stably expressed on activated dendritic cells. 1532 Aug 71