Uyama Toru, Kitagawa Hiroshi, Tanaka Junko, Tamura Jun-ichi, Ogawa Tomoya, Sugahara Kazuyuki
Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan.
J Biol Chem. 2003 Jan 31;278(5):3072-8. doi: 10.1074/jbc.M209446200. Epub 2002 Nov 13.
We identified a novel human chondroitin N-acetylgalactosaminyltransferase, designated chondroitin GalNAcT-2 after a BLAST analysis of the GenBank(TM) data base using the sequence of a previously described human chondroitin N-acetylgalactosaminyltransferase (chondroitin GalNAcT-1) as a probe. The new cDNA sequence contained an open reading frame encoding a protein of 542 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 60% identity to that of human chondroitin GalNAcT-1. Like chondroitin GalNAcT-1, the expression of a soluble form of the protein in COS-1 cells produced an active enzyme, which not only transferred beta1,4-N-acetylgalactosamine (GalNAc) from UDP-[(3)H]GalNAc to a polymer chondroitin representing growing chondroitin chains (beta-GalNAc transferase II activity) but also to GlcUA beta 1-3Gal beta 1-O-C(2)H(4)NHCbz, a synthetic substrate for beta-GalNAc transferase I that transfers the first GalNAc to the core tetrasaccharide in the protein-linkage region of chondroitin sulfate. In contrast, the tetrasaccharide serine (GlcUA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser) derived from the linkage region, which is an inert acceptor substrate for chondroitin GalNAcT-1, served as an acceptor substrate. The coding region of this enzyme was divided into seven discrete exons, which is similar to the genomic organization of the chondroitin GalNAcT-1 gene, and was localized to chromosome 10q11.22. Northern blot analysis revealed that the chondroitin GalNAcT-2 gene exhibited a ubiquitous but differing expression in human tissues, and the expression pattern differed from that of chondroitin GalNAcT-1. Thus, we demonstrated redundancy in the chondroitin GalNAc transferases involved in the biosynthetic initiation and elongation of chondroitin sulfate, which is important for understanding the biosynthetic mechanisms leading to the selective chain assembly of chondroitin/dermatan sulfate on the linkage region tetrasaccharide common to various proteoglycans containing chondroitin/dermatan sulfate and heparin/heparan sulfate chains.
我们鉴定出一种新型人类软骨素N - 乙酰半乳糖胺基转移酶，在使用先前描述的人类软骨素N - 乙酰半乳糖胺基转移酶（软骨素GalNAcT - 1）的序列作为探针，对GenBank™数据库进行BLAST分析后，将其命名为软骨素GalNAcT - 2。新的cDNA序列包含一个开放阅读框，编码一个含有542个氨基酸的蛋白质，具有II型跨膜蛋白拓扑结构。该氨基酸序列与人类软骨素GalNAcT - 1的氨基酸序列具有60%的同一性。与软骨素GalNAcT - 1一样，该蛋白的可溶性形式在COS - 1细胞中的表达产生了一种活性酶，它不仅能将UDP - [(3)H]GalNAc中的β1,4 - N - 乙酰半乳糖胺（GalNAc）转移到代表正在生长的软骨素链的聚合物软骨素上（β - GalNAc转移酶II活性），还能转移到GlcUAβ1 - 3Galβ1 - O - C(2)H(4)NHCbz上，GlcUAβ1 - 3Galβ1 - O - C(2)H(4)NHCbz是β - GalNAc转移酶I的一种合成底物，β - GalNAc转移酶I将第一个GalNAc转移到硫酸软骨素蛋白连接区的核心四糖上。相比之下，来自连接区的四糖丝氨酸（GlcUAβ1 - 3Galβ1 - 3Galβ1 - 4Xylβ1 - O - Ser），它是软骨素GalNAcT - 1的惰性受体底物，却充当了受体底物。该酶的编码区被分为七个离散的外显子，这与软骨素GalNAcT - 1基因的基因组组织相似，并且定位于染色体10q11.22。Northern印迹分析显示，软骨素GalNAcT - 2基因在人类组织中呈现普遍但不同的表达，其表达模式与软骨素GalNAcT - 1不同。因此，我们证明了参与硫酸软骨素生物合成起始和延伸的软骨素GalNAc转移酶存在冗余，这对于理解导致在含有硫酸软骨素/硫酸皮肤素和肝素/硫酸乙酰肝素链的各种蛋白聚糖共有的连接区四糖上选择性组装软骨素/硫酸皮肤素链的生物合成机制很重要。
