Differential roles of two N-acetylgalactosaminyltransferases, CSGalNAcT-1, and a novel enzyme, CSGalNAcT-2. Initiation and elongation in synthesis of chondroitin sulfate.
By a tblastn search with beta 1,4-galactosyltransferases as query sequences, we found an expressed sequence tag that showed similarity in beta 1,4-glycosyltransferase motifs. The full-length complementary DNA was obtained by a method of 5'-rapid amplification of complementary DNA ends. The predicted open reading frame encodes a typical type II membrane protein comprising 543 amino acids, the sequence of which was highly homologous to chondroitin sulfate N-acetylgalactosaminyltransferase (CSGalNAcT-1), and we designated this novel enzyme CSGalNAcT-2. CSGalNAcT-2 showed much stronger N-acetylgalactosaminyltransferase activity toward glucuronic acid of chondroitin poly- and oligosaccharides, and chondroitin sulfate poly- and oligosaccharides with a beta 1-4 linkage, i.e. elongation activity for chondroitin and chondroitin sulfate, but showed much weaker activity toward a tetrasaccharide of the glycosaminoglycan linkage structure (GlcA-Gal-Gal-Xyl-O-methoxyphenyl), i.e. initiation activity, than CSGalNAcT-1. Transfection of the CSGalNAcT-1 gene into Chinese hamster ovary cells yielded a change of glycosaminoglycan composition, i.e. the replacement of heparan sulfate on a syndecan-4/fibroblast growth factor-1 chimera protein by chondroitin sulfate, however, transfection of the CSGalNAcT-2 gene did not. The above results indicated that CSGalNAcT-1 is involved in the initiation of chondroitin sulfate synthesis, whereas CSGalNAcT-2 participates mainly in the elongation, not initiation. Quantitative real-time PCR analysis revealed that CSGalNAcT-2 transcripts were highly expressed in the small intestine, leukocytes, and spleen, however, both CSGalNAcTs were ubiquitously expressed in various tissues.
Sato
Takashi
T
Glycogene Function Team, Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Open Space Laboratory C-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
Gotoh
Masanori
M
Kiyohara
Katsue
K
Akashima
Tomohiro
T
Iwasaki
Hiroko
H
Kameyama
Akihiko
A
Mochizuki
Hideo
H
Yada
Toshikazu
T
Inaba
Niro
N
Togayachi
Akira
A
Kudo
Takashi
T
Asada
Masahiro
M
Watanabe
Hideto
H
Imamura
Toru
T
Kimata
Koji
K
Narimatsu
Hisashi
H
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002809:Chondroitin Sulfates / Q000096:biosynthesis*; D006224:Cricetinae; D017931:DNA Primers; D018076:DNA, Complementary; D005784:Gene Amplification; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D002809:Chondroitin Sulfates; D017350:N-Acetylgalactosaminyltransferases; C472559:chondroitin sulfate N-acetylgalactosaminyltransferase-1; C472560:chondroitin sulfate N-acetylgalactosaminyltransferase-2; C023220:(N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase