Human thrombomodulin: complete cDNA sequence and chromosome localization of the gene

Abstract

A human umbilical vein endothelial cell cDNA library in lambda gt11 was screened for expression of thrombomodulin antigens with affinity-purified rabbit polyclonal anti-thrombomodulin immunoglobulin G (IgG) and mouse monoclonal anti-human thrombomodulin IgG. Among 7 million recombinant clones screened, 12 were recognized by both antibodies. Two of these, lambda HTm10 and lambda HTm12, were shown to encode thrombomodulin by comparison of the amino acid sequence deduced from the nucleotide sequence to the amino acid sequence determined directly from tryptic peptides of thrombomodulin. Thrombomodulin mRNA was estimated to be 3.7 kilobases in length by Northern blot analysis of endothelial cell and placental poly(A)+ RNA. Thrombomodulin mRNA was not detected in human brain, HepG2 hepatoma cells, or the monocytic U937 cell line. Additional cDNA clones were selected by hybridization with the 1.2-kilobase insert of lambda HTm10. One isolate, lambda HTm15, contained a 3693 base pair cDNA insert with an apparent 5'-noncoding region of 146 base pairs, an open reading frame of 1725 base pairs, a stop codon, a 3'-noncoding region of 1779 base pairs, and a poly(A) tail of 40 base pairs. The cDNA sequence encodes a 60.3-kDa protein of 575 amino acids. The predicted protein sequence includes a signal peptide of approximately 21 amino acids, an amino-terminal ligand-binding domain of approximately 223 amino acids, an epidermal growth factor (EGF) homology region of 236 amino acids, a serine/threonine-rich segment of 34 amino acids, a membrane-spanning domain of 23 amino acids, and a cytoplasmic tail of 38 amino acids. The EGF-homology region consists of six tandemly repeated EGF-like domains.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutant oocytic low density lipoprotein receptor gene family member causes atherosclerosis and female sterility.

“…The synthesized cDNA was used for subsequent PCR amplifications. The following oligonucleotides, P1-7, were used for PCR primers: P1, 5'-CGACGGGATATCAGGAA-GATTGGCC-3'; P2, 5'-AGGAAGAACAGCCCAAGCTC-CTGCT-3'; P3, 5'-ACCCTAGTAAACAACCTCAATGATG-3'; P4, 5'-TGGAGGAAGTCTTTCAGCCACAAGC-3'; P5, 30 cycles. To amplify the sequence of the 5' end of the receptor mRNA, 5' rapid amplification of cDNA ends was carried out with primer P7 and the 5' rapid amplification of cDNA ends system (GIBCO/ BRL).…”

Cloning and Subcellular Location of an Arabidopsis Receptor-Like Protein That Shares Common Features with Protein-Sorting Receptors of Eukaryotic Cells

“…Cys-rich domains and / or EGF-like repeats have been found in the yeast vacuolar sorting receptor VpslOp (Marcusson et al, 1994), the human EGFR and LDLRs and their related proteins (Herz et al, 1988), and the thrombomodulin receptor (Wen et al, 1987). The Cys-rich domains in the VpslO protein may be involved in ligand binding (Horazdovsky et al, 1995), whereas in the LDLR they are believed to bind its ligands and apolipoproteins B and E .…”

Expression of Thrombomodulin in Atherosclerotic Lesions and Mitogenic Activity of Recombinant Thrombomodulin in Vascular Smooth Muscle Cells

“…2 The EGF-like domain consists of 6 tandem EGF-like motifs that are homologous to domain III of the human EGF precursor. 3 These EGF-like structures are associated with the cofactor activity of TM during thrombin-dependent protein C activation. 4,5 Soluble TM fragments containing the EGF-like domain of native cellular TM 6 have been detected in circulating plasma.…”