Sarkar M, Schachter H
The Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Biol Chem. 2001 Feb;382(2):209-17. doi: 10.1515/BC.2001.028.
A TBLASTN search of the Drosophila melanogaster expressed sequence tag (EST) database with the amino acid sequence of human UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I, EC 2.4.1.101) as probe yielded a clone (GM01211) with 56% identity over 36 carboxy-terminal amino acids. A 550 base pair (bp) probe derived from the EST clone was used to screen a Drosophila cDNA library in lambda-ZAP II and two cDNAs lacking a start ATG codon were obtained. 5'-Rapid amplification of cDNA ends (5'-RACE) yielded a 2828 bp cDNA containing a full-length 1368 bp open reading frame encoding a 456 amino acid protein with putative N-terminal cytoplasmic (5 residues) and hydrophobic transmembrane (20 residues) domains. The protein showed 52% amino acid sequence identity to human GnT I. This cDNA, truncated to remove the N-terminal hydrophobic domain, was expressed in the baculovirus/Sf9 system as a secreted protein containing an N-terminal (His)6 tag. Protein purified by adsorption to and elution from nickel beads converted Man alpha1-6(Man alpha1-3)Man beta-octyl (M3-octyl) to Man alpha1-6(GlcNAc beta1-2Man alpha1-3)Man beta-octyl. The Km values (0.7 and 0.03 mM for M3-octyl and UDP-GlcNAc respectively), temperature optimum (37 degrees C), pH optimum (pH 5 to 6) and divalent cation requirements (Mn > Fe, Mg, Ni > Ba, Ca, Cd, Cu) were similar to mammalian GnT I. TBLASTN searches of the Berkeley Drosophila Genome Project database with the Drosophila GnT I cDNA sequence as probe allowed localization of the gene to chromosomal region 2R; 57A9. Comparison of the cDNA and genomic DNA sequences allowed the assignment of seven exons and six introns; all introns showed GT-AG splice site consensus sequences. This is the first insect GnT I gene to be cloned and expressed.
以人UDP-N-乙酰葡糖胺：α-3-D-甘露糖苷β-1,2-N-乙酰葡糖胺基转移酶I（GnT I，EC 2.4.1.101）的氨基酸序列为探针，对黑腹果蝇表达序列标签（EST）数据库进行TBLASTN搜索，得到一个克隆（GM01211），其36个羧基末端氨基酸的同一性为56%。从EST克隆中获得的一个550碱基对（bp）的探针用于筛选λ-ZAP II中的果蝇cDNA文库，得到两个缺少起始ATG密码子的cDNA。5'-cDNA末端快速扩增（5'-RACE）得到一个2828 bp的cDNA，其包含一个全长1368 bp的开放阅读框，编码一个456个氨基酸的蛋白质，该蛋白质具有推定的N末端胞质结构域（5个残基）和疏水跨膜结构域（20个残基）。该蛋白质与人类GnT I的氨基酸序列同一性为52%。将该cDNA截短以去除N末端疏水结构域，在杆状病毒/Sf9系统中作为含有N末端（His）6标签的分泌蛋白进行表达。通过吸附到镍珠上并从镍珠上洗脱纯化的蛋白质将Manα1-6（Manα1-3）Manβ-辛酯（M3-辛酯）转化为Manα1-6（GlcNAcβ1-2Manα1-3）Manβ-辛酯。其Km值（M3-辛酯和UDP-GlcNAc分别为0.7和0.03 mM）、最适温度（37℃）、最适pH（pH 5至6）和二价阳离子需求（Mn＞Fe、Mg、Ni＞Ba、Ca、Cd、Cu）与哺乳动物GnT I相似。以果蝇GnT I cDNA序列为探针，对伯克利果蝇基因组计划数据库进行TBLASTN搜索，可将该基因定位到染色体区域2R；57A9。通过比较cDNA和基因组DNA序列，确定了7个外显子和6个内含子；所有内含子均显示GT-AG剪接位点共有序列。这是第一个被克隆和表达的昆虫GnT I基因。
