Glycobiology Advance Access published online on May 25, 2005
Glycobiology, doi:10.1093/glycob/cwi082
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1 Department of Biochemistry, Sasaki Institute, 2-2, Kanda-Surugadai, Chiyoda-ku, Tokyo 101- 0062, and CREST, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama, Japan
* To whom correspondence should be addressed. We characterized a novel member of the
Received February 23, 2005
Revised May 18, 2005
Accepted May 19, 2005
Article
Characterization of a Novel Galactose:
1,3-N-Acetylglucosaminyltransferase (3Gn-T8): The Complex Formation of 3Gn-T2 and 3Gn-T8 Enhances Enzymatic Activity
2 Department of Biochemistry, Sasaki Institute, 2-2, Kanda-Surugadai, Chiyoda-ku, Tokyo 101- 0062
Katsuko Yamashita, E-mail: yamashita{at}sasaki.or.jp
Abstract 
1,3-N-acetylglucosaminyltransferase (3Gn-T) gene family, 3Gn-T8. A recombinant soluble form of 3Gn-T8 was expressed in Pichia pastoris, and its substrate specificity was compared to that of 3Gn-T2. The two enzymes had similar substrate specificities, and recognized tetra-antennary N-glycans and 2,6-branched triantennary glycans in preference to 2,4-branched triantennary glycans, biantennary glycans, and lacto-N-neotetraose, indicating their specificity for 2,6-branched structures such as [Gal1
4GlcNAc12(Gal14GlcNAc16)Man
16Man]. Interestingly, when soluble recombinant 3Gn-T2 and 3Gn-T8 were mixed, the Vmax/Km value of the mixture was 9.3- and 160-fold higher than those of individual 3Gn-T2 and -T8, respectively. Sephacryl S-300 gel filtration of the enzymes revealed that apparent molecular weights of each 3Gn-T2, 3Gn-T8, and the mixture were 90-160 kDa, 45-65 kDa, and 110-210 kDa, respectively, suggesting that 3Gn-T2 and -T8 can form a complex with enhanced enzymatic activity. This is the first report demonstrating that in vitro mixed glycosyltransferases show enhanced enzymatic activity through formation of a heterocomplex. These results suggested that 3Gn-T8 and 3Gn-T2 are cooperatively involved in elongation of specific branch structures of multi-antennary N-glycans.
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