Two specific N-acetylglucosaminyltransferases, α-1,3-mannoside:β-2-N-acetylglucosaminyltransferase (transferase I) and α-1,6-mannoside:β-2-N-acetylglucosaminyltransferase (transferase II), which catalyze the transfer of N-acetylglucosamine (GlcNAc) from uridine diphospho-GlcNAc to terminal branched α-mannosyl (Man) residues, were purified from liver metastases of human colon adenocarcinoma. Transferase I was assayed with Manα1,6(Manα1,3)Manα1,6(Manα1,3)Manβ1,4GlcNAcβ1, 4GlcNAc-Asn (Km 0.35 mm), and transferase II was assayed with Manα1,6(GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4-GlcNAc-Asn (Km 1.0 mm), in which Asn is asparagine. The Km of transferase I for Manα1,6(Manα1,3)Manβ1,4GlcNAc-β1,4)-(Fucα1,6)GlcNAc-Asn was 1 mm. The specificity of the interaction of transferase I with ovalbumin, ovomucoid, the modified heavy chain of porcine immunoglobulin G and glycopeptides prepared from these glycoproteins was examined by kinetic and structural analysis. The best macromolecular substrates for transferase I were ovalbumin devoid of terminal GlcNAc and some mannose, a solubilized preparation of the heavy chain of porcine immunoglobulin G, devoid of sialic acid, galactose, and terminal GlcNAc, and untreated ovomucoid. The apparent Kms were 45, 19, and 390 µm for ovalbumin, the modified heavy chain of immunoglobulin G, and untreated ovomucoid, respectively. The apparent Km of the enzyme for uridine diphospho-GlcNAc was not significantly influenced by the nature of the glycoprotein acceptor, and it varied between 14 and 20 µm for the different glycoproteins. The structures of the oligosaccharide chains in these glycoproteins which acted as acceptors for the purified enzyme were determined. A major glycopeptide product with the structure Manα1,3(Manα1,6)Manα1,6(14C-GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAc-β1,4-GlcNAc-Asn was isolated from both ovalbumin and ovomucoid following incubation with transferase I. The specificity of the enzyme for terminal branched mannosyl residues attached to a β-linked mannose unit greatly restricts the action of this transferase to this juncture in the synthesis of complex-type oligosaccharide chains of N-asparagine-linked glycoproteins.

Two α-mannosidases, one specific for the cleavage of α-1,2-linked mannose (α-mannosidase I) and the other cleaving both α1,3- and α1,6-linked mannoses (α-mannosidase II), were also partially purified from microsome fractions of human liver metastases. α-Mannosidase I was assayed with [2,6-3H]Manlabeled glycopeptide, Manα1,2Manα1,6(Manα1,2Manα1,3)Manα1,6(Manα1,2Manα1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc-Asn, and α-mannosidase II was assayed with Manα1,6(Manα1,3)Manα1,6(GlcNAcβ1,2Manα1,3)Manβ1,4-GlcNAcβ1,4GlcNAc-Asn (Km, 0.2 mm). The specificity and kinetic properties of the α-mannosidases and β-1,2-N-acetylglucosaminyltransferases described in the present report provide a mechanism for the specific elongation of high-mannose oligosaccharide chains to complex-type oligosaccharides found in carcinoembryonic antigen.


This investigation was supported by USPHS Grant CA23703 awarded by the National Cancer Institute, Department of Health and Human Services.

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