Escrivá H, Pierce A, Coddeville B, González F, Benaissa M, Léger D, Wieruszeski J M, Spik G, Pamblanco M
Departament de Bioquímica i Biologia Molecular, Facultat de Ciències Biològiques, Universitat de València, Spain.
Biochem J. 1995 Apr 1;307 ( Pt 1)(Pt 1):47-55. doi: 10.1042/bj3070047.
The complete cDNA for rat mammary-gland transferrin (Tf) has been sequenced and also the native protein isolated from milk in order to analyse the structure of the main glycan variants present. A lactating-rat mammary-gland cDNA library in lambda gt10 was screened with a partial cDNA copy of rat liver Tf and subsequently rescreened with 5' fragments of the longest clones. This produced a 2275 bp insert coding for an open reading frame of 695 amino acid residues. This includes a 19-amino acid signal sequence and the mature protein containing 676 amino acids and one N-glycosylation site in the C-terminal domain at residue 490. Phylogenetic analysis was carried out using 14 translated Tf nucleotide sequences, and the derived evolutionary tree shows that at least three gene duplication events have occurred during Tf evolution, one of which generated the N- and C-terminal domains and occurred before separation of arthropods and chordates. The two halves of human melanotransferrin are more similar to each other than to any other sequence, which contrasts with the pattern shown by the remaining sequences. Native rat milk Tf is separated into four bands on native PAGE that differ only in their sialic acid content: one biantennary glycan is present containing either no sialic acid residues or up to three. The complete structures of the two major variants were determined by methylation, m.s. and 400 MHz 1H-n.m.r. spectroscopy. They contain either one or two neuraminic acid residues (alpha 2-->6)-linked to galactose in conventional biantennary N-acetyl-lactosamine-type glycans. Most contain fucose (alpha 1-->6)-linked to the terminal non-reducing N-acetylglucosamine.
已对大鼠乳腺转铁蛋白（Tf）的完整cDNA进行了测序，并从乳汁中分离出天然蛋白，以分析其中主要聚糖变体的结构。用大鼠肝脏Tf的部分cDNA拷贝筛选λgt10中的泌乳大鼠乳腺cDNA文库，随后用最长克隆的5'片段进行再筛选。这产生了一个2275 bp的插入片段，编码一个由695个氨基酸残基组成的开放阅读框。其中包括一个19个氨基酸的信号序列和成熟蛋白，该成熟蛋白含有676个氨基酸，在第490位残基的C末端结构域中有一个N-糖基化位点。使用14个翻译后的Tf核苷酸序列进行了系统发育分析，所得进化树表明，在Tf进化过程中至少发生了三次基因重复事件，其中一次产生了N末端和C末端结构域，发生在节肢动物和脊索动物分离之前。人黑素转铁蛋白的两半彼此之间比与任何其他序列更相似，这与其余序列所示的模式形成对比。天然大鼠乳Tf在天然PAGE上被分离成四条带，它们仅在唾液酸含量上有所不同：存在一种双天线聚糖，其要么不含唾液酸残基，要么最多含三个唾液酸残基。通过甲基化、质谱和400 MHz 1H-核磁共振光谱确定了两种主要变体的完整结构。它们在传统的双天线N-乙酰乳糖胺型聚糖中含有一个或两个与半乳糖（α2→6）相连的神经氨酸残基。大多数含有与末端非还原N-乙酰葡糖胺（α1→6）相连的岩藻糖。
