A Necessary and Sufficient Determinant for Protein-selective Glycosylation in Vivo

Erin Miller,Dorothy Fiete,Nicquet M.J Blake,Mary Beranek,Edward L Oates,Yiling Mi,Daniel S Roseman,Jacques U Baenziger

doi:10.1074/jbc.m708160200

Abstract

A limited number of glycoproteins including luteinizing hormone and carbonic anhydrase-VI (CA6) bear N-linked oligosaccharides that are modified with beta1,4-linked N-acetylgalactosamine (GalNAc). The selective addition of GalNAc to these glycoproteins requires that the beta1,4-N-acetylgalactosaminyltransferase (betaGT) recognize both the oligosaccharide acceptor and a peptide recognition determinant on the substrate glycoprotein. We report here that two recently cloned betaGTs, betaGT3 and betaGT4, that are able to transfer GalNAc to GlcNAc in beta1,4-linkage display the necessary glycoprotein specificity in vivo. Both betaGTs transfer GalNAc to N-linked oligosaccharides on the luteinizing hormone alpha subunit and CA6 but not to those on transferrin (Trf). A single peptide recognition determinant encoded in the carboxyl-terminal 19-amino acid sequence of bovine CA6 mediates transfer of GalNAc to each of its two N-linked oligosaccharides. The addition of this 19-amino acid sequence to the carboxyl terminus of Trf confers full acceptor activity onto Trf for both betaGT3 and betaGT4 in vivo. The complete 19-amino acid sequence is required for optimal GalNAc addition in vivo, indicating that the peptide sequence is both necessary and sufficient for recognition by betaGT3 and betaGT4.

Highlights

Does not itself comprise the site of modification can be added to a structurally unrelated glycoprotein and confer selective modification of this unrelated glycoprotein
We reported that the basic amino acids within the sequence PLRSKK that is present in the pituitary glycoprotein hormone ␣ subunit are critical elements of the peptide recognition determinant conferring transfer of GalNAc to N-linked oligosaccharides in vitro [26, 27]
The N-linked oligosaccharides on CA6(Wt) expressed in HEK 293T cells are modified with ␤1,4-linked GalNAc, suggesting that, like the glycoprotein hormone ␣ subunit, CA6(Wt) has a recognition determinant that results in its selective modification with GalNAc when expressed by HEK 293T cells

Summary

Introduction

Does not itself comprise the site of modification can be added to a structurally unrelated glycoprotein and confer selective modification of this unrelated glycoprotein. We reported that the basic amino acids within the sequence PLRSKK that is present in the pituitary glycoprotein hormone ␣ subunit are critical elements of the peptide recognition determinant conferring transfer of GalNAc to N-linked oligosaccharides in vitro [26, 27]. By expressing these chimeric glycoproteins in cells that express either ␤GT3 or ␤GT4, we were able to quantitatively compare their extent of modification with ␤1,4-linked GalNAc in vivo Using this approach we show that ␤GT3 and ␤GT4 are proteinspecific, recognizing a peptide determinant in the protein substrate to allow efficient and protein-selective transfer of GalNAc to their oligosaccharides. We have identified a 19-amino acid sequence that is both necessary and sufficient to mediate protein-specific GalNAc addition by ␤GT3 and ␤GT4 The addition of this sequence to a glycoprotein that is not recognized by either ␤GT3 or ␤GT4 converts the glycoprotein into one that is selectively modified with ␤1,4-linked GalNAc in vivo

Methods

Results

Conclusion