Oligosaccharyltransferase (OST), an integral component of the endoplasmic-reticulum membrane, catalyses the transfer of dolichyl diphosphate-linked oligosaccharides to specific asparagine residues forming part of the Asn-Xaa-Thr/Ser sequence. We have studied the binding and catalytic properties of the enzyme from pig liver using peptide analogues derived from the acceptor peptide N-benzoyl-Asn-Gly-Thr-NHCH3 by replacing either asparagine or threonine with amino acids differing in size, stereochemistry, polarity and ionic properties. Acceptor studies showed that analogues of asparagine and threonine with bulkier side chains impaired recognition by OST. Reduction of the beta-amide carbonyl group of asparagine yielded a derivative that, although not glycosylated, was strongly inhibitory (50% inhibition at approximately 140 microM). This inhibition may be due to ion-pair formation involving the NH3+ group and a negatively charged base at the active site. Hydroxylation of asparagine at the beta-C position increased Km and decreased Vmax, indicating an effect on both binding and catalysis. The threo configuration at the beta-C atom of the hydroxyamino acid was essential for substrate binding. A peptide derivative obtained by replacement of the threonine beta-hydroxy group with an NH2 group was found to display acceptor activity. This shows that the primary amine is able to mimic the hydroxy group during transglycosylation. The pH optimum with this derivative is shifted by approximately 1 pH unit towards the basic region, indicating that the neutral NH2 group is the reactive species. The various data are discussed in terms of the catalytic mechanism of OST, particular emphasis being placed on the role of threonine/serine in increasing the nucleophilicity of the beta-amide of asparagine through hydrogen-binding.

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A crucial step in plant xanthone biosynthesis is the cyclization of an intermediate benzophenone to a xanthone. In cultured cells of Centaurium erythraea RAFN, 2,3¢,4,6-tetrahydroxybenzophenone (THBP) was shown to be intramolecularly coupled to 1,3,5-trihydroxyxanthone, whereas in cell cultures of Hypericum androsaemum L. it was coupled to form the isomeric 1,3,7-trihydroxyxanthone. These regioselective cyclizations that occur ortho and para, respectively, to the 3¢-hydroxy group of the benzophenone depend on cytochrome 450 , as shown by the eectiveness of established 450 inhibitors and blue-light-reversible carbon monoxide inhibition. Furthermore, the reactions absolutely require NADPH and O 2 . The underlying reaction mechanism is probably an oxidative phenol coupling that is catalyzed regioselectively by xanthone synthases. These enzymes are proposed to be cytochrome 450 oxidases. The intramolecular cyclizations of THBP to 1,3,5-and 1,3,7-trihydroxyxanthones catalyzed by the two xanthone synthases represent an important branch point in the plant xanthone biosynthetic pathway.Abbreviations: HMBC = heteronuclear multiple-bond connectivity; HMQC = heteronuclear multiple-quantum coherence; NMR = nuclear magnetic resonance; THBP = 2,3¢,4,6-tetrahydroxybenzophenone

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.

The mouse hemoglobin-derived decapeptide Hb (67-76), VITAFNEGLK, which binds well to Ek and is non-immunogenic in CBA/J mice, was O-glycosylated with the tumor-associated carbohydrate Tn (alpha-D-N-acetylgalactosamine, or alpha-D-GalNAc). Each of the ten positions in the peptide was substituted with serine or threonine having the Tn antigen attached. The complete set of Tn-glycosylated peptides were then studied for binding to Ek and for immunogenicity in CBA/J mice. All of those glycopeptides which had the Tn attached to serine or threonine at a position in the peptide where, according to the crystal structure determinations, the amino acid side chain was oriented downwards into the binding site of the major histocompatibility complex (MHC) molecule, completely lost their capacity for binding to Ek. This was the case for the glycopeptides with Tn attached at position 68 and 76, which are the major anchor residues and for those with Tn attached at position 71 and 73, which function as secondary anchor residues. Those glycopeptides which had Tn attached to serine or threonine at positions where the side chain pointed away from the binding site maintained their capacity for binding to Ek, except for those with Tn attached at position 70 and 74. Furthermore, some of the MHC-binding glycopeptides were immunogenic. In particular, this was the case for the glycopeptide with Tn attached to the central position 72 in the decapeptide. From previous studies, this is known to be the dominant T cell receptor contact residue of Hb (67-76). The results suggest that T cells may be capable of recognizing epitopes which are partially defined by a small glycan group.