Three types of unusual post-translational modification have been found within conserved amino acid sequences in epidermal growth factor homology regions (EGF modules) of some multidomain proteins. beta-Hydroxyaspartate and beta-hydroxyasparagine are found within -Cys-Xxx-Asp/Asn-Xxx-Xxx-Xxx-Xxx-Tyr/Phe-Xxx-Cys-Xxx-Cys- sequences. (Xyl alpha 1-->3)Xyl alpha 1-->3Glc beta 1-->O-Ser glycans at conserved sites within -Cys-Xxx-Ser-Xxx-Pro-Cys- sequences have been reported in several proteins. Fuc alpha 1-->O-Thr/Ser modifications have been found at conserved sites within -Cys-Xxx-Xxx-Gly-Gly-Thr/Ser-Cys- sequences. More recently, it has been discovered that the Ser residue corresponding to the potential O-fucosylation site in human factor IX carries the novel tetrasaccharide NeuAc alpha 2-->6Gal beta 1-->4GlcNAc beta 1-->3Fuc alpha 1-->O-Ser; this tetrasaccharide can be considered to be an extension of the Fuc alpha 1-->O moiety. The consensus sequences for these post-translational modifications are in close proximity to each other; e.g. human factor IX has all three unusual modifications within a 12 amino acid linear sequence. In proteins with multiple EGF modules, the O-glycosidic modifications have been found only within the N-terminal EGF module; beta-hydroxyaspartate/asparagine residues are not restricted in the same fashion. Little is known yet about the functions of, or possible relationships between, any of these modifications.

The present paper describes the structures of the N-linked oligosaccharides of the human-immunodeficiency-virus (HIV) envelope glycoprotein gp120 (cloned from the HTLV-III B isolate and expressed as a secreted fusion protein after transfection of Chinese-hamster ovary cells), which is known to bind with high affinity to human T4-lymphocytes. Oligosaccharides were released from peptide by hydrazinolysis, fractionated by paper electrophoresis, high-performance lectin-affinity chromatography and Bio-Gel P-4 column chromatography, and their structures determined by sequential exoglycosidase digestions in conjunction with methylation analysis. The glycoprotein was found to be unique in its diversity of oligosaccharide structures. These include high-mannose type and hybrid type, as well as four categories of complex-type chains: mono-, bi-, tri- and tetra-antennary, with or without N-acetyl-lactosamine repeats, and with or without a core-region fucose residue. Among the sialidase-treated oligosaccharides, no less than 29 structures were identified as follows: (formula; see text) where G is galactose, GN is N-acetylglucosamine, M is mannose, F is fucose, and '+/- ' means that residues are present in a proportion of chains. The actual number of oligosaccharide structures is much greater, since before desialylation there was evidence that, among the hybrid and complex-type chains, all but 6% contained sialic acid at the C-3 position of terminal galactose residues, and partially sialylated forms of the bi- and multi-antennary chains were present. Detailed evidence for the proposed oligosaccharide sequences will be published as a supplementary paper [T. Mizuochi, M. W. Spellman, M. Larkin, J. Solomon, L. J. Basa & T. Feizi (1988) Biomed. Chromatogr., in the press].

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A GDP-fucose:polypeptide fucosyltransferase was purified 5000-fold to homogeneity from Chinese hamster ovary cell extracts in the absence of detergent. The purification procedure included two affinity chromatographic steps using the acceptor substrate, a recombinant factor VII EGF-1 domain, and the donor substrate analog, GDP-hexanolamine, as ligands. The purified enzyme migrates as a single band of 44,000 daltons on SDS-polyacrylamide gel electrophoresis and is itself a glycoprotein with more than one high mannose type oligosaccharide chain with a total molecular weight of 4000. The K m values for factor VII EGF-1 domain and GDP-fucose are 15 and 6 M, respectively. The V max is 2.5 mol⅐min ؊1 ⅐mg ؊1 . The presence of 50 mM Mn 2؉ increased the enzyme activity 17-fold, but Mn 2؉ was not absolutely required, since the enzyme exhibited some activity even in the presence of EDTA. The acceptor substrate specificity was studied using site-directed mutagenesis of human factor IX EGF domain. Only one of several differently folded species could serve as acceptor substrate, although they all had the same molecular weight as determined by liquid chromatography on-line with mass spectrometry. This indicates that the enzyme requires proper folding of the epidermal growth factor domain for its activity.The identification of O-linked fucose attached to a specific protein was first made by Kentzer et al. (1), who found a residue of fucose covalently linked through an O-glycosidic bond to the EGF 1 domain of recombinant urokinase. The same modification was later identified to be on tissue plasminogen activator (2), human factor VII (3), human factor XII (4), and a plasminogen activator from the saliva of a vampire bat (5). Similar fucosylation was also shown to occur on the EGF domain of human factor IX (6), but in this case the fucose residue was at the reducing end of a tetrasaccharide: NeuAc␣236Gal␤134GlcNAc␤133Fuc␣13O-Ser 61 (7). In all cases described above, the attachment of O-linked fucose to serine or threonine was found to occur on EGF domains within the sequence Cys-Xaa-Xaa-Gly-Gly-Ser/Thr-Cys (8).Although all examples of mammalian O-fucosylation characterized to date have been modifications of EGF domains (8), it is possible that O-linked fucosylation occurs in non-EGF-containing proteins. Studies by two other laboratories using the lec1 mutant cell line indicated that O-fucosylation occurs on many proteins in Chinese hamster ovary cells (9, 10). These studies were not performed in a manner that would enable EGF domain-containing proteins to be distinguished from those without EGF domains. Structural analysis has also revealed that O-linked fucose is present on an insect protease inhibitor (11), although the flanking amino acids were not represented by the consensus sequence described above, and the protease inhibitor sequence is not homologous to an EGF domain.We have focused our research mainly on the biosynthesis of this post-translational modification. Recently, we reported the development of an assay for the GDP-...