Zhang G S, Mehringer J H, Van Deerlin V M, Kozak C A, Tollefsen D M
Division of Hematology-Oncology, Washington University School of Medicine, St Louis, Missouri 63110.
Biochemistry. 1994 Mar 29;33(12):3632-42. doi: 10.1021/bi00178a021.
Heparin cofactor II (HCII) is a glycoprotein in human plasma that inhibits thrombin rapidly in the presence of dermatan sulfate or heparin. Unexpectedly, we found that HCII activity in murine plasma is present in two proteins of 68 and 72 kDa. The two proteins have the same N-terminal amino acid sequence, and both react with an antibody raised against the C-terminal nine amino acid residues of murine HCII predicted from the cDNA sequence. Treatment of the two proteins with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase yields a single 54-kDa band. Thus, murine plasma contains two forms of HCII that appear to have identical amino acid sequences but differ in the composition of their N-linked oligosaccharides. HCII cDNA clones isolated from a murine liver library include a 1434 bp open reading frame following the first Met codon, a TAA stop codon, and 580 bp of 3'-untranslated sequence terminating in a poly(A) tail. The amino acid sequence deduced from the cDNA contains the N-terminal sequence of purified murine plasma HCII preceded by a 23-residue hydrophobic sequence presumed to be the signal peptide. The amino acid sequence of murine HCII is 87% identical to that of human HCII, the greatest variability occurring in the N-terminal portion of the protein. Northern blot analysis reveals a 2.3-kb HCII mRNA in murine and human liver, but no HCII mRNA is detectable in heart, brain, spleen, lung, skeletal muscle, kidney, testis, placenta, pancreas, or intestine. Southern blot analysis of restriction fragment length polymorphisms in progeny on interspecific and intersubspecific crosses indicates that mice have a single HCII gene (designated Hcf2), which maps to chromosome 16 between Prm-1 and Igl. The murine HCII gene is approximately 7.1 kb in size and consists of at least four exons and three introns. The intron/exon organization is identical to that of the human HCII gene except at the 5' end, where the murine gene may lack a large intron in the 5'-untranslated region. Our results indicate that HCII is more highly conserved than the human and murine homologues of other serpins such as alpha 1-antitrypsin and alpha 1-antichymotrypsin.
肝素辅因子II（HCII）是人类血浆中的一种糖蛋白，在硫酸皮肤素或肝素存在的情况下能迅速抑制凝血酶。出乎意料的是，我们发现小鼠血浆中的HCII活性存在于两种分别为68 kDa和72 kDa的蛋白质中。这两种蛋白质具有相同的N端氨基酸序列，并且都能与针对根据cDNA序列预测的小鼠HCII C端九个氨基酸残基产生的抗体发生反应。用肽-N4-（N-乙酰-β-葡糖胺基）天冬酰胺酶处理这两种蛋白质会产生一条单一的54 kDa条带。因此，小鼠血浆中含有两种形式的HCII，它们似乎具有相同的氨基酸序列，但N-连接寡糖的组成不同。从小鼠肝脏文库中分离出的HCII cDNA克隆在第一个Met密码子之后包含一个1434 bp的开放阅读框、一个TAA终止密码子以及580 bp的3'非翻译序列，该序列以聚腺苷酸尾结束。从cDNA推导的氨基酸序列包含纯化的小鼠血浆HCII的N端序列，其前面有一个推测为信号肽的23个残基的疏水序列。小鼠HCII的氨基酸序列与人类HCII的氨基酸序列有87%的同一性，最大的变异性出现在该蛋白质的N端部分。Northern印迹分析显示在小鼠和人类肝脏中有一条2.3 kb的HCII mRNA，但在心脏、大脑、脾脏、肺、骨骼肌、肾脏、睾丸、胎盘胰腺或肠道中未检测到HCII mRNA。对种间和亚种间杂交后代的限制性片段长度多态性进行Southern印迹分析表明，小鼠有一个单一的HCII基因（命名为Hcf2），它定位于16号染色体上Prm-1和Igl之间。小鼠HCII基因大小约为7.1 kb，由至少四个外显子和三个内含子组成。除了5'端外，内含子/外显子的组织方式与人类HCII基因相同，在5'端，小鼠基因在5'-非翻译区可能缺少一个大的内含子。我们的结果表明，与其他丝氨酸蛋白酶抑制剂如α1-抗胰蛋白酶和α1-抗糜蛋白酶的人类和小鼠同源物相比，HCII的保守性更高。
