UDPgalactose:glycoprotein–N‐acetyl‐d‐galactosamine 3‐β‐d‐galactosyltransferase activity synthesizing O‐glycan core 1 is controlled by the amino acid sequence and glycosylation of glycopeptide substrates

Abstract

In order to investigate the role of the peptide moiety of glycoproteins in the control of 0-glycan biosynthesis, UDPga1actose:glycoprotein-N-acetyl-D-galactosamine 3-P-D-galactosyltransferase (core 1 P3-Gal-T) from rat liver was tested for its specificity towards GalNAc-containing glycopeptide substrates. Series of glycopeptides have been synthesized by solid-phase synthesis, protected with an acetyl group on the amino terminal and an amide group on the carboxy terminal, based on variations of the repeat sequences of human intestinal mucin. Most glycopeptides were excellent substrates for core 1 /33-Gal-T compared to benzyl a-D-galactosamine as indicated by their relatively high V,,,,IK,,,. The enzyme preferred threonine a-D-galactosamine Thr(Ga1NAc) to serine a-D-galactosamine. Pro on the carboxy-terminal side adjacent to Thr(Ga1NAc) was inhibitory. Negatively charged amino acids on either side showed a low K,; substrates with negatively charged amino acids on the amino-terminal side were highly efficient substrates, suggesting charge-charge interactions between enzyme and substrate. GalPl-3GalNAca residues adjacent to Thr(Ga1NAc) reduced the activity. Product analysis using glycopeptide substrates with three adjacent GalNAc residues showed incorporation of one, two and a small amount of three Gal residues per molecule with an uneven distribution of the potential di-galactosylated isomers. These studies indicate that, in addition to initial glycosylation, the second step in the glycosylation pathways of 0-glycans is also controlled by the structure and glycosylation of the peptide core of substrates.

Determination of the Site-specific Oligosaccharide Distribution of the O-Glycans Attached to the Porcine Submaxillary Mucin Tandem Repeat

“…where the specificities of both the ppGalNAc and core 1 transferases were proposed to be affected by steric effects of neighboring glycosylated residues (22,23,32,33,58,59).…”

“…Depending on the initial and subsequent substitutions on the GalNAc residue, a wide range of O-linked core structures is possible (25). Little, however, is known of the effects of local peptide sequence on O-glycan elongation (23,(32)(33)(34)(35)(36)(37), although a number of examples of site-specific elongation of N-linked glycans are known (30, 38 -46). Factors affecting site-specific N-glycan 1 The abbreviations used are: ppGalNAc transferase, UDP-GalNAc: polypeptide ␣-GalNAc transferase; ␣2-Fuc transferase, GDP-fucose: galactose ␤-1,3-␣1,2-fucosyltransferase; core 1 ␤3-Gal transferase, UDP-galactose:␣-N-acetylgalactosamine ␤-1,3-galactosyltransferase; ST6GalNAc, GalNAc ␣2,6-sialyltransferase; Fuc, fucose; NeuNGl, N-glycoloylneuraminic acid or sialic acid; PSM, porcine submaxillary mucin; TR, tandem repeat; PTH, phenylthiohydantoin; Native GalNAc or (T)TTTR-PSM, Core 1 or (T)TROTR-PSM, Trisaccharide, or (T)TROGNTR-PSM, a series of differentially deglycosylated preparations of the PSM multiple tandem repeat domain described under "Materials and Methods" and under "Results," where (T) represents the 81-residue tandem repeat domain obtained after trypsinolysis; TFMSA, trifluoromethanesulfonic acid; SOG, glycosylated Ser; SOH, nonglycosylated Ser; TOG, glycosylated Thr; TOH, nonglycosylated Thr.…”

Detection of O-glycosylated proteins from different Trichinella species muscle larvae total extracts

“…This observation could be explained by the fact that the specificity of this enzyme is controlled by the amino acid sequence of the peptide substrates (Granovsky et al, 1994). Obviously, sialyl-Tn and sialyl-T do not exist in Trichinella spp.…”

“…Moreover, alcian-blue staining of T. spiralis muscle larvae tissue sections and histochemistry with the lectins specific for sialic acid also did not show any labeling (Milcheva et al, 2009). The absence of particularly α6-silaylation in T. spiralis muscle larvae ESP has been reported by Gruden-Movsesijan et al (2002). We used HPA in the context of its ability to recognize Tnantigen (Roth, 1984), a feature that resembles the VVL-B 4 affinity ( Table 1).…”

“…The glycobiology of Trichinella is becoming a subject of rising interest. N-glycosylation has been characterized in detail in adult T. spiralis total extracts (Morelle et al, 2000) and also in ESP from T. spiralis muscle larvae (Gruden-Movsesijan et al, 2002), in which the presence of the unusual sugar tyvelose has been discovered (Wisnewski et al, 1993;Reason et al, 1994). Except for one report (Gruden-Movesijan et al, 2002), there is virtually no information available on the structure and the synthesis of O-glycans in Trichinella.…”