Expression cloning of cDNA encoding a human β-1,3-N-acetylglucosaminyltransferase that is essential for poly-N-acetyllactosamine synthesis

Abstract

The structure and biosynthesis of poly-Nacetyllactosamine display a dramatic change during development and oncogenesis. Poly-N-acetyllactosamines are also modified by various carbohydrate residues, forming functional oligosaccharides such as sialyl Le x . Herein we describe the isolation and functional expression of a cDNA encoding ␤-1,3-N-acetylglucosaminyltransferase (iGnT), an enzyme that is essential for the formation of poly-Nacetyllactosamine. For this expression cloning, Burkitt lymphoma Namalwa KJM-1 cells were transfected with cDNA libraries derived from human melanoma and colon carcinoma cells. Transfected Namalwa cells overexpressing the i antigen were continuously selected by f luorescenceactivated cell sorting because introduced plasmids containing Epstein-Barr virus replication origin can be continuously amplified as episomes. Sibling selection of plasmids recovered after the third consecutive sorting resulted in a cDNA clone that directs the increased expression of i antigen on the cell surface. The deduced amino acid sequence indicates that this protein has a type II membrane protein topology found in almost all mammalian glycosyltransferases cloned to date. iGnT, however, differs in having the longest transmembrane domain among glycosyltransferases cloned so far. The iGnT transcript is highly expressed in fetal brain and kidney and adult brain but expressed ubiquitously in various adult tissues. The expression of the presumed catalytic domain as a fusion protein with the IgG binding domain of protein A enabled us to demonstrate that the cDNA encodes iGnT, the enzyme responsible for the formation of GlcNAc␤1 3 3Gal␤1 3 4GlcNAc 3 R structure and poly-N-acetyllactosamine extension.

Poly-N-acetyllactosamine Extension inN-Glycans and Core 2- and Core 4-branchedO-Glycans Is Differentially Controlled by i-Extension Enzyme and Different Members of the β1,4-Galactosyltransferase Gene Family

“…The cDNA encoding iGnT was cloned into pcDNA3.1, resulting in pcDNA3.1-iGnT as described previously (29). pcDNAI-A, harboring cDNA encoding a signal sequence and an IgG binding domain of Staphylococcus aureus protein A, was constructed as described before (32).…”

“…pcDNAI-A, harboring cDNA encoding a signal sequence and an IgG binding domain of Staphylococcus aureus protein A, was constructed as described before (32). The catalytic domain of iGnT was cloned into this vector, resulting in pcDNAI-A⅐iGnT (29).…”

“…pcDNAI-A and pcDNAI-A⅐iGnT were separately transfected with LipofectAMINE Plus (Life Technologies) into COS-1 cells as described previously (29). The chimeric enzyme released into serum-free OPTI-MEM was used after adsorbing the protein A chimeric enzyme to IgG-Sepharose 6FF (Amersham Pharmacia Biotech) as described previously (33).…”

“…To this end, we have previously demonstrated (27) that poly-N-acetyllactosamines in core 2-branched O-glycans are synthesized with a newly discovered member of ␤1,4-galactosyltransferase, ␤4Gal-TIV (28) and a newly cloned i-extension enzyme (iGnT) (29). We have also found that ␤4Gal-TI, iGnT, and I-branching enzyme (IGnT) are involved in the synthesis of I-branched poly-Nacetyllactosamines (30).…”

Tumor suppressor function of laminin-binding α-dystroglycan requires a distinct β3- N -acetylglucosaminyltransferase

“…LARGE displays 2 distinct structural domains homologous to UDP-glucose protein glucosyltransferase (14) and ␤3-Nacetylglucosaminyltransferase 1 (␤3GnT1) (11). ␤3GnT1 was originally identified by expression cloning as an enzyme that synthesizes i-antigen, a linear poly-N-acetyllactosamine, on human fetal red blood cell (15). ␤3GnT1 is widely distributed in various tissues and is structurally distinct from the other members of ␤3GnT gene family, and it was proposed that ␤3GnT1 might functionally differ from those enzymes (16).…”