S2.3 Cloning and identification of cDNAS encoding human?1?6N-acetylglucosaminyltransferases

Abstract

UDP-N-acetylglucosamine:a-6-D-mannoside f3-1,2-N-acetylglucosaminyltransferase II (GnT II, EC 2.4.1.143) is a Golgi enzyme catalyzing an essential step in the conversion of high mannose to complex N-glycans. A 1.2 kb probe from a rat liver cDNA encoding GnT II (D'Agostaro et al., this volume) was used to screen a human genomic DNA library in iEMBL3 prepared from human leukocyte DNA partially digested with Sau3A (Clontech). Several positive clones were obtained of which four were plaque purified. Restriction endonuclease digests with SstI, EcoRI and XbaI, followed by Southern blot analysis with the 1.2 kb rat probe, gave a different pattern for each of the four phage clones. Hybridizing fragments (3.0 and 3.5 kb) were isolated from two of the phage clones (HG30 and HG36, respectively) and subcloned into pBlueScript (priG30 and priG36). Sequencing revealed that the inserts in priG30 and priG36 are overlapping clones containing 5.5 kb of genomic DNA. pr iG30 contains a 1341 bp open reading frame encoding a 447 amino acid protein, 250 bp of GC-rich 5 ' upstream sequence and 1.4 kb of 3 '-downstream sequence. priG36 encodes 2.75 kb of 5 '-upstream sequence and 750 bp of the 5' -end of the open reading frame. The protein sequence showed the domain structure typical of all previously cloned glycosyltransferases, i.e., a short nine amino acid N-terminal cytoplasmic domain, a 22 amino acid hydrophobic noncleavable signal-anchor transmembrane domain and a 416 amino acid C-terminal catalytic domain. Northern analyses of several human lymphocyte lines showed a message size at 2.6 kb. There is no sequence homology to any previously cloned gtycosyltransferase including human fJ-1,2-N-acetylglucosaminyttransferase I (GnT I) which has 445 amino acids with a 416 amino acid catalytic domain. The entire coding regions of human and mouse GnT I and of human and rat GnT II are on single exons. The human GnT I gene (MGAT1) has been localized to chromosome 5q35 whereas the human GriT II gene (MGAT2) is on chromosome 14q21; localizations were done by hybridization and PCR analyses of genomic DNA from hybrid cell lines and by fluorescent in situ hybridization (FISH) using genomic DNA probes. There is a 90% identity between the amino acid sequences of the catalytic domains of human and rat GnT II (see Petrarca et al., this volume, for data on rat GnT II).