Primary binding domain of bovine von Willebrand factor fragment expressed in E. coli.

Bovine vWF cDNA has been cloned from a bovine endothelial cell library. A fragment of this cDNA, corresponding to amino acid sequence Leu 469-Ser 723, called primary adhesion domain (PAD-1), and containing the binding sites for platelet glycoprotein Ib (GPIb), heparin and collagen, has been expressed in E. coli. The reduced and alkylated form of fragment PAD-1 inhibited native vWF binding to GPIb. Fragment PAD-1 bound to heparin and botrocetin in a specific and dose dependent manner as did the native vWF. In a solid-phase assay, fragment PAD-1 bound to calf skin collagen in contrast to a human vWF recombinant fragment (Ser 445-Val 733) which was inactive in the same assay. The studies presented in this paper demonstrated that the A1 domain of bovine vWF contained the GPIb, heparin, botrocetin as well as collagen binding sites and that integrity of the disulfide bond (Cys 509-Cys 695), did not seem to be essential for binding of bovine vWF fragment to GPIb.

Comparison of the 5'-flanking sequences of the human and bovine von Willebrand factor-encoding genes reveals alternation of highly homologous domains with species-specific Alu-type repeats.

von Willebrand factor (vWF), a multimeric glycoprotein important for hemostasis, is specifically synthesized in endothelial cells and in platelet precursors (megakaryocytes). Recent studies from two laboratories, including ours, were published regarding the cell-specific transcription of reporter genes controlled by the human (hu) vWF promoter in transfected bovine (bo) endothelial cells and cells of non-endothelial origins. In order to verify that the regulatory domains previously characterized in the 5' region of hu vWF are also present in bo vWF, we have sequenced 1.9 kb upstream from the cap site, plus five exons. The comparison of human and bovine exons two to five shows homology of 83% at the nucleotide (nt) level and 78% at the deduced amino-acid sequence level. The bovine and human exons one, which are non-coding and span 233 and 250 bp, respectively, are only 64% homologous. In the first exon, potentially involved in endothelial-cell-specific transcription, the binding site for factor Sp1 is present in bo vWF, whereas the GATA sequence is replaced by a GACA sequence. The sequence corresponding to the human basal promoter, located between nt -89 and +19, is well conserved with 82% homology. However, the human TAATTA sequence (at nt -32) considered to be a TATA box, is replaced by TCATTA, and the CCAAT element at nt -18 is replaced by CCTGT. Among domains involved in transcription, the negative regulatory domain located 5' from the core promoter is highly conserved. The bovine sequence upstream from the first intron can be aligned with the human sequence up to nt -656 which is located in a polymorphic poly(GT)18-26 sequence. At this site, the bovine DNA contains an insertion of 523 bp which corresponds to a bovine Alu-type art2 repeat of 331 bp flanked by bovine microsatellites. The art2 sequence is an Alu-type repeat in artiodactyls with at least 100,000 copies in the bovine genome. Upstream from this insertion, 368 bp of the bovine sequence can be aligned with the human counterpart up to a 9-bp element which flanks an human Alu repeat which is absent from the bovine DNA. Upstream of the human Alu insertion and a duplicate of the 9-bp element, the two sequences are again homologous.

PCR to identify specific clones of interest for DNA sequencing.

We describe a rapid method for identifying specific clones of interest for the purpose of sequencing. The method essentially is polymerase chain reaction using one internal primer and one vector specific primer. The procedure is particularly useful when relatively large numbers of clones are to be examined either to establish the nucleotide sequence of a full-length cDNA or to find a specific section of a large DNA. The relative orientations of inserts in different clones can also be determined using the same procedure.

Sequencing of the primary adhesion domain of bovine von Willebrand factor.

A cDNA library, constructed from bovine heart endothelial cell poly(A)+ RNA, was screened using a BstXI fragment of human von Willebrand and factor (vWF) cDNA as a probe. This probe codes for the major adhesion domain of vWF that includes the GPIb, collagen and heparin binding domains. Of the ten positive clones obtained, a clone that spanned the region of interest was sequenced by the dideoxynucleotide method yielding a sequence of 1550 bp. This region of the bovine cDNA codes for amino acids corresponding to #262 to #777 in human vWF and encompasses the entire pro adhesion domain. Both the nucleotide sequence and the deduced amino acid sequence are 82% homologous to those of human vWF. Cysteine residues #471, 474, 509 and 695, which form intrachain bonds in human vWF, are also present in the bovine vWF sequence.

Complete cDNA sequence of bovine alpha 1-antitrypsin.

A cDNA clone coding for the entire bovine alpha 1-antitrypsin molecule has been isolated from a lambda gt11 bovine liver cDNA library using a human alpha 1-antitrypsin cDNA as a probe. The bovine cDNA was sequenced by the dideoxynucleotide chain termination method. Comparison of the translated amino acid sequence of the bovine alpha 1-antitrypsin with those of the human, baboon, sheep, rat and mouse demonstrates the preservation of most of the critical structural determinants. The bovine and the sheep molecules have a sequence homology of 94% and both the molecules contain four cysteine residues; there is only one cysteine in the others.

Proteolytic and immunologic comparison of human and bovine von Willebrand factor.

The structures of bovine and human vWF were compared by proteolysis with Staphylococcus aureus V8 protease and rattlesnake venom Protease I. Fragments were analyzed for chain composition, heparin binding, collagen binding, platelet agglutinating activity and recognition by a panel of monoclonal antibodies which reacted with both bovine and human vWF. Similar large fragments from the C-terminal domain of vWF were seen in each case. The N-terminal domain resulting from cleavage of bovine vWF was much smaller than that seen upon digestion of human vWF with V8 protease. Protease I destroyed the heparin binding domain in human vWF. Bovine vWF was much less sensitive to proteolysis than was human vWF.