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N-glycosylation of human interferon-gamma: glycans at Asn-25 are critical for protease resistance.

ABSTRACT: Human interferon-gamma (IFN-gamma) is a secretory, dimeric glycoprotein that forms a compact globular structure with potential N-linked glycosylation sites at Asn-25 and Asn-97 on the surface of the dimer. In natural leucocyte IFN-gamma (nIFN-gamma), 52%, 39% and 9% of the monomers are core-glycosylated in two, one or none of the potential N-glycosylation sites respectively. Chemical cross-linking of nIFN-gamma with glutaraldehyde revealed that 4, 3, 2 or 1 glycosylation sites occupied 28%, 40%, 26% and 6% of the dimers respectively. In baculovirus-produced wild-type (Wt) and N-linked glycosylation site-defective mutant (N25Q or N97Q, Asn-25 or Asn-97 substituted by Gln) IFN-gamma proteins, the extent of core glycosylation of monomers reflected the glycan composition of dimers. This suggests that dimers are formed randomly and independently of glycosylation. The glycan residues of IFN-gamma, especially at Asn-25, play an important role in protease resistance. Unglycosylated recombinant IFN-gamma proteins (from Escherichia coli and baculovirus) and N25Q IFN-gamma were sensitive to crude granulocyte protease, purified elastase, cathepsin G and plasmin degradation. Fully glycosylated nIFN-gamma and baculovirus Wt and N97Q IFN-gamma showed full or partial resistance to these proteases. These results emphasize the importance of glycan residues, especially at Asn-25, in the proteolytic stability of human IFN-gamma. Whether the differential glycosylation of n- and recombinant IFN-gamma (rIFN-gamma) is reflected in their biological activities in tissues or their clinical applicability is not known.

SUBMITTER: Sareneva T

PROVIDER: S-EPMC1136836 | biostudies-other | 1995 May

REPOSITORIES: biostudies-other

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Project description:Follicle-stimulating hormone (FSH) is a gonadotrope-derived heterodimeric glycoprotein. Both the common α- and hormone-specific β subunits contain Asn-linked N-glycan chains. Recently, macroheterogeneous FSH glycoforms consisting of β-subunits that differ in N-glycan number were identified in pituitaries of several species and subsequently the recombinant human FSH glycoforms biochemically characterized. Although chemical modification and in vitro site-directed mutagenesis studies defined the roles of N-glycans on gonadotropin subunits, in vivo functional analyses in a whole-animal setting are lacking. Here, we have generated transgenic mice with gonadotrope-specific expression of either an HFSHB(WT) transgene that encodes human FSHβ WT subunit or an HFSHB(dgc) transgene that encodes a human FSHβ(Asn7Δ 24Δ) double N-glycosylation site mutant subunit, and separately introduced these transgenes onto Fshb null background using a genetic rescue strategy. We demonstrate that the human FSHβ(Asn7Δ 24Δ) double N-glycosylation site mutant subunit, unlike human FSHβ WT subunit, inefficiently combines with the mouse α-subunit in pituitaries of Fshb null mice. FSH dimer containing this mutant FSHβ subunit is inefficiently secreted with very low levels detectable in serum. Fshb null male mice expressing HFSHB(dgc) transgene are fertile and exhibit testis tubule size and sperm number similar to those of Fshb null mice. Fshb null female mice expressing the mutant, but not WT human FSHβ subunit-containing FSH dimer are infertile, demonstrate no evidence of estrus cycles, and many of the FSH-responsive genes remain suppressed in their ovaries. Thus, HFSHB(dgc) unlike HFSHB(WT) transgene does not rescue Fshb null mice. Our genetic approach provides direct in vivo evidence that N-linked glycans on FSHβ subunit are essential for its efficient assembly with the α-subunit to form FSH heterodimer in pituitary. Our studies also reveal that N-glycans on FSHβ subunit are essential for FSH secretion and FSH in vivo bioactivity to regulate gonadal growth and physiology.

Project description:Recombinant human interferon-gamma (Hu-IFN-gamma) produced by Chinese-hamster ovary (CHO) cells was analysed by immunoprecipitation and SDS/PAGE. Up to twelve molecular-mass variants were secreted by this cell line. Three variants were recovered after enzymic removal of all N-linked oligosaccharides or when glycosylation was inhibited by tunicamycin. The presence of three polypeptide forms rather than a single form suggested that proteolytic cleavage had occurred at two sites in both the glycosylated and non-glycosylated forms. Proteolytically cleaved IFN-gamma was more prevalent in cell lysates than in the secreted glycoprotein. In common with naturally produced IFN-gamma, both fully glycosylated IFN-gamma (asparagine residues 28 and 100 occupied) and partially glycosylated product (thought to be substituted at position Asn28) were secreted. This was deduced from the Mr of the glycosylated products and the relative amounts of sialic acid expressed by each variant. In contrast with naturally produced IFN-gamma, non-glycosylated IFN-gamma was also secreted by the transfected CHO cells. When the cells were grown in batch culture in serum-free medium under pH and dissolved-oxygen control, the proportion of non-glycosylated IFN-gamma increased from 3 to 5% after 3 h, to 30% of the total IFN-gamma present after 195 h. This change in the proportion of glycosylated protein produced was not seen when metabolically labelled IFN-gamma was incubated for 96 h with cell-free supernatant from actively growing CHO cells. This implied that an alteration in intracellular glycosylation was occurring rather than a degradation of oligosaccharide side chains after secretion. The decrease in IFN-gamma glycosylation was independent of the glucose concentration in the culture medium, but could be related to specific growth and IFN-gamma production rates, as these declined steadily after 50 h of culture, in line with the increased production of non-glycosylated IFN-gamma.

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N-glycosylation of human interferon-gamma: glycans at Asn-25 are critical for protease resistance.

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