Peptide-specific Transfer of N-Acetylgalactosamine to O-Linked Glycans by the Glycosyltransferases β1,4-N-Acetylgalactosaminyl Transferase 3 (β4GalNAc-T3) and β4GalNAc-T4

Dorothy Fiete,Mary Beranek,Jacques U Baenziger

doi:10.1074/jbc.m112.371880

Dorothy Fiete, Mary Beranek + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m112.371880

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Abstract

N- and O-linked oligosaccharides on pro-opiomelanocortin both bear the unique terminal sequence SO(4)-4-GalNAcβ1,4GlcNAcβ. We previously demonstrated that protein-specific transfer of GalNAc to N-linked oligosaccharides on glycoprotein substrates is dependent on the presence of both an oligosaccharide acceptor and a peptide recognition motif consisting of a cluster of basic amino acids. We characterized how two β1,4-N-acetylgalactosaminyltransferases, β4GalNAc-T3 and β4GalNAc-T4, require the presence of both the peptide recognition motif and the N-linked oligosaccharide acceptors to transfer GalNAc in β1,4-linkage to GlcNAc in vivo and in vitro. We now show that β4GalNAc-T3 and β4GalNAc-T4 are able to utilize the same peptide motif to selectively add GalNAc to β1,6-linked GlcNAc in core 2 O-linked oligosaccharide structures to form Galβ1,3(GalNAcβ1,4GlcNAcβ1,6)GalNAcαSer/Thr. The β1,4-linked GalNAc can be further modified with 4-linked sulfate by either GalNAc-4-sulfotransferase 1 (GalNAc-4-ST1) (CHST8) or GalNAc-4-ST2 (CHST9) or with α2,6-linked N-acetylneuraminic acid by α2,6-sialyltransferase 1 (ST6Gal1), thus generating a family of unique GalNAcβ1,4GlcNAcβ (LacdiNAc)-containing structures on specific glycoproteins.

Highlights

LacdiNAc (GalNAc␤1,4GlcNAc) is present on O- and N-linked carbohydrate moieties of pro-opiomelanocortin
The presence of LacdiNAc-modified N-linked oligosaccharides on murine POMC [2, 3] raised the possibility that the addition of ␤1,4-linked GalNAc to the ␤1,6linked GlcNAc in O-linked oligosaccharides with a core 2 structure is mediated by the same ␤1,4N-acetylgalactosaminyltransferases (␤4GalNAc-T) that we have reported to account for protein-specific transfer of GalNAc to N-linked oligosaccharides on glycoproteins such as the luteinizing hormone (LH) (4 –9) and carbonic anhydrase-6 (CA6) [10]
The CTP sequence was added to the carboxyl terminus of GLuc (Gaussia luciferase) followed by the 19-amino acid sequence from CA6 (CA1–19) that we have shown is recognized by ␤4GalNAc-T3 and ␤4GalNAc-T4 followed by the Myc-His epitope tag

Summary

Background

LacdiNAc (GalNAc␤1,4GlcNAc) is present on O- and N-linked carbohydrate moieties of pro-opiomelanocortin. The presence of LacdiNAc-modified N-linked oligosaccharides on murine POMC [2, 3] raised the possibility that the addition of ␤1,4-linked GalNAc to the ␤1,6linked GlcNAc in O-linked oligosaccharides with a core 2 structure (see Fig. 1, structure 4) is mediated by the same ␤1,4N-acetylgalactosaminyltransferases (␤4GalNAc-T) that we have reported to account for protein-specific transfer of GalNAc to N-linked oligosaccharides on glycoproteins such as the luteinizing hormone (LH) (4 –9) and carbonic anhydrase-6 (CA6) [10]. GalNAc Transfer to O-Linked Oligosaccharides rides terminating LacdiNAc modified with sulfate or NeuNAc are recognized by the mannose receptor (18 –23) and the asialoglycoprotein receptor [24, 25], respectively, and regulate the circulatory half-lives of glycoprotein hormones bearing these structures in vivo. LacdiNAc termini modified with sulfate or NeuNAc on O-linked structures may be recognized by the same and/or additional receptors and have important biological consequences in vivo

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DISCUSSION