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K Nehrke

Publications and source records attributed to K Nehrke.

27 records · Page 2Linked to original sources
PubMed1998

Isoform-specific O-glycosylation by murine UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-T3, in vivo.

Multiple isoforms of UDP-GalNAc:polypeptide N-acetylgalactosaminyl- transferase (ppGaNTase) have been cloned and expressed from a variety of organisms. In general, these isoforms display different patterns of tissue-specific expression, but exhibit overlapping substrate specificities, in vitro . A peptide substrate, derived from the sequence of the V3 loop of the HIV gp120 protein (HIV peptide), has previously been shown to be glycosylated in vitro exclusively by the ppGaNTase-T3 (Bennett et al. , 1996). To determine if this isoform-specificity is maintained in vivo , we have examined the glycosylation of this substrate when it is expressed as a reporter peptide (rHIV) in a cell background (COS7 cells) which lacks detectable levels of the ppGaNTase-T3. Glycosylation of rHIV was greatly increased by coexpression of a recombinant ppGaNTase-T3. Overexpression of ppGaNTase-T1 yielded only partial glycosylation of the reporter. We have also determined that the introduction of a proline residue at the +3 position flanking the potential glycosylation site eliminated ppGaNTase-T3 selectivity toward rHIV observed both in vivo and in vitro .

Amino Acid Sequence↗
PubMed1997

Biosynthesis of a low-molecular-mass rat submandibular gland mucin glycoprotein in COS7 cells.

We have examined the biosynthesis of a low-molecular-mass mucin from rat submandibular gland (RSMG) expressed recombinantly in COS7 tissue culture cells, focusing primarily on the addition of carbohydrate to the protein core of the mucin. We find evidence for N-linked glycosylation, but this modification is not required for secretion of the mucin. Similarly, although the recombinant RSMG mucin, like its native counterpart, contains large amounts of O-linked carbohydrate, chain extension beyond the initial O-linked GalNAc moiety is not required for secretion. We have identified partially glycosylated mucin by a combination of metabolic pulse-chase and lectin precipitations of the biosynthetic intermediates. Our results suggest that the addition of GalNAc to threonine and serine in the RSMG mucin does not occur simultaneously, as has been described for other O-glycosylated proteins.

Acetylgalactosamine↗
PubMed1997

Charge distribution of flanking amino acids inhibits O-glycosylation of several single-site acceptors in vivo.

From surveys of known O-glycosylation sites and in vitro glycosylation assays with synthetic peptide acceptors, it appears that the presence of charged amino acids near serine/threonine residues reduces the likelihood of O-glycosylation by UDP-GalNAc polypeptide:N-acetylgalactosaminyltransferases (ppGaNTases). Previously, we demonstrated that the in vivo O-glycosylation of a sequence derived from a known glycosylation site of human von Willebrand factor (PHMAQVTVGPGL) was markedly reduced when charged residues were substituted at position -1 and +3 relative to the single threonine. In contrast, acidic residues at positions -2, +1, and +2 had no effect (Nehrke et al., 1996), suggesting that charge distribution but not charge density was important. To determine whether the charge distribution effect on O-glycosylation is limited to a specific sequence context or restricted to unique isoforms of ppGaNTase, we have analyzed the in vivo O-glycosylation of six secreted recombinant reporter proteins in three different cell backgrounds. The differential presence of known ppGaNTase transcripts was determined in each cell type by Northern blot analysis. Each reporter, which contains a single site of O-glycosylation, was O-glycosylated in a cell-background-specific manner; digestion with O-glycanase and alpha-N-acetylgalactosaminidase following mild acid hydrolysis suggested that simple type II core structures were acquired. However, in COS7 cells, one reporter peptide acquired glycosaminoglycans in preference to mucin-type O-glycans. Regardless of cell background or the reporter examined, the substitution of glutamic acid residues at positions -1 and +3 markedly diminished the level of mucin-type O-glycosylation. Charge distribution would appear, therefore, to play a more general role in determining the extent to which solitary O-glycosylation sites are modified.

Amino Acid Sequence↗
PubMed1996

Charge distribution of flanking amino acids influences O-glycan acquisition in vivo.

The elements that regulate O-glycosylation are poorly understood. We have developed a novel in vivo system to analyze the role of flanking sequence on the modification of a single well characterized O-glycosylation site derived from human von Willebrand factor (PHMAQVTVGPGL). A secreted chimeric reporter protein, containing the human von Willebrand factor sequence, an antibody recognition epitope, and a heart muscle kinase site, was engineered and expressed in COS7 and MCF-7 cells. Glycosylated and non-glycosylated forms of the immunoprecipitated reporter were resolved electrophoretically and their relative amounts quantitated. Using mutational analysis we find that the glycosylation apparatus of COS7 cells can accommodate a broad range of changes in the flanking sequence without compromising glycosylation, but that the distribution of charged amino acids flanking the O-glycosylation site can have a profound influence on glycosylation with position -1 relative to the glycosylation site being particularly sensitive. A combination of acidic residues at positions -1 and +3 almost completely eliminates glycosylation of the reporter in both COS7 and MCF-7 cells. The overall density of charged amino acids is less important since substitution of acidic residues at position -2, +1, and +2 had no effect in the level of glycosylation observed.

Amino Acid Sequence↗