Expression of variant von Willebrand factor (vWF) cDNA in heterologous cells: requirement of the pro-polypeptide in vWF multimer formation.

Von Willebrand factor (vWF) is a multimeric plasma glycoprotein synthesized by vascular endothelial cells as a pre-pro-polypeptide with a highly repetitive domain structure, symbolized by the formula: (H)-D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-(OH) A heterologous expression system for the synthesis of recombinant vWF protein was developed, consisting of a monkey kidney cell line (COS-1), transfected with full-length vWF cDNA. This system was shown to mimic the constitutive secretory pathway of vWF in endothelial cells, since dimerization and multimerization occur similarly. To determine whether the pro-polypeptide, composed of the domains D1 and D2, is involved in vWF multimerization, a vWF cDNA was constructed that lacked the coding sequence for the pro-polypeptide. The mutant vWF protein, expressed by COS-1 cells transfected with this cDNA, did not assemble beyond the dimer stage. From this observation, we conclude that (i) dimerization does not involve the pro-polypeptide of pro-vWF and (ii) the presence of the pro-polypeptide, as part of pro-vWF, is obligatory for multimerization. It is argued that the interactions, required for interchain binding, are mediated by the D domains.     

Domains involved in multimer assembly of von willebrand factor (vWF): multimerization is independent of dimerization.

The precursor protein of von Willebrand factor (pro-vWF) consist of four repeated domains, denoted D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2. The domains D1 and D2 constitute the amino-terminal pro-polypeptide and the remaining domains mature vWF, generated upon proteolytic processing. We have shown previously that the pro-polypeptide of pro-vWF is obligatory for assembly of pro-vWF dimers into multimers, a process vital for efficient adhesion of platelets to an injured vessel wall. Here, we have employed full length vWF cDNA to construct a series of deletion mutants, based on the homology between the various domains. Specifically, the domains D', D3 or both were deleted and the multimeric pattern of the mutant vWF proteins was analysed after transient expression in COS-1 cells. It is demonstrated that in addition to the pro-polypeptide, both the D' and the D3 domain are required for multimer assembly. Furthermore, by analysing a construct containing only the domains D' and D3 next to the pro-polypeptide it is shown that this is the only part of the vWF protein involved in multimer assembly. Since, the formation of pro-vWF dimers relies on the carboxy-terminal area of mature vWF, it is concluded that multimer assembly is a process independent of dimerization.     

Biogenesis of von Willebrand factor-containing organelles in heterologous transfected CV-1 cells.

Von Willebrand factor (vWF) is a multimeric protein involved in the adhesion of platelets to an injured vessel wall. vWF is synthesized by the endothelial cell and the megakaryocyte as a precursor protein (pro-vWF) that consists of four repeated domains, denoted D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2. Previously, we have defined the domains on the pro-vWF molecule involved in dimerization as well as the domains involved in multimer assembly of vWF dimers. In the endothelial cell, part of the vWF multimers is stored in specialized organelles, the Weibel-Palade bodies. By using immunoelectron microscopy, we demonstrate that upon expression of full-length vWF cDNA, vWF-containing organelles are encountered in monkey kidney CV-1 cells that are morphologically similar to the endothelial-specific Weibel-Palade bodies. Expression in CV-1 cells of a series of vWF cDNA deletion mutants, lacking one or more domains, revealed that only those vWF mutant proteins that are able to assemble into multimers are encountered in dense-cored vesicles. Our data show that this process is independent of a particular domain on vWF and indicate that a 'condensed', multimeric vWF is required for targeting to the Weibel-Palade body.     

Assembly and routing of von Willebrand factor variants: the requirements for disulfide-linked dimerization reside within the carboxy-terminal 151 amino acids

The precursor protein of von Willebrand factor (pro-vWF) consists of four different repeated domains, denoted D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2, followed by a carboxy-terminal region of 151 amino acids without obvious internal homology. Previously, we have shown the requirement of the domains D1, D2, D', and D3 of pro-vWF in the assembly of pro-vWF dimers into multimers. Here, we define the domains of vWF involved in dimerization, using deletion mutants of full-length vWF cDNA transiently expressed in monkey kidney COS-1 cells. It is shown that only the carboxy-terminal 151 amino acid residues of vWF are required for dimerization. In addition, by analyzing a construct, encoding only the carboxy-terminal 151 amino acids of vWF, we find that the formation of dimers is an event independent of other domains present on pro-vWF, such as the domains C1 and C2 previously suggested to be involved in dimerization. Furthermore, it is shown that a deletion mutant of vWF, lacking the carboxy-terminal 151 amino acid residues and thus unable to dimerize, is proteolytically degraded in the ER. In contrast, a mutant protein, composed only of the carboxy-terminal 151 amino acids of vWF, and able to dimerize, is transported from the ER in a similar fashion as wild-type vWF. The role of the ER in the assembly of vWF is discussed with regard to the data presented in this paper on the intracellular fate of several vWF mutant proteins.     

Construction and characterization of an active factor VIII variant lacking the central one-third of the molecule

The primary structure of factor VIII consists of 2332 amino acids that exhibit 3 distinct structural domains, including a triplicated region (A domains), a unique region of 909 amino acids (B domain), and a carboxy-terminal duplicated region (C domains), that are arranged in the order A1-A2-B-A3-C1-C2. The B domain (residues 741-1648) of factor VIII is lost when factor VIII is activated by thrombin, which proteolytically processes factor VIII to active subunits of Mr 50,000 (domain A1), 43,000 (domain A2), and 73,000 (domains A3-C1-C2). To determine if the B domain is required for factor VIII coagulant activity, a variant was constructed by using recombinant DNA techniques in which residues 797-1562 were eliminated. This shortened the B domain from 909 to 142 amino acids. This variant factor VIIIdes-797-1652 was expressed in mammalian cells and was found to be functional. The factor VIIIdes-797-1562 protein was purified and shown to be processed by thrombin in the same manner as full-length factor VIII. The factor VIIIdes-797-1562 variant also bound to von Willebrand factor (vWF) immobilized on Sepharose. These results indicate that most of the highly glycosylated B domain of factor VIII is not required for the expression of factor VIII coagulant activity and its interaction with vWF.     

Proteolytic cleavage of the precursor of von Willebrand factor is not essential for multimer formation

Monkey kidney cells (COS-1), transfected with full-length human von Willebrand factor (vWF) cDNA encoding the precursor of vWF (pro-vWF), mimic the characteristics of the biosynthesis and of the constitutive secretory pathway, displayed by cultured vascular endothelial cells. Such heterologous transfected cells are able to cleave pro-vWF, generating the propolypeptide and mature vWF, and to assemble pro-vWF dimers into a series of multimers, similarly to endothelial cells. Evidence is presented showing that proteolytic processing of pro-vWF by COS-1 cells occurs at the peptide bond between arginine and serine in the sequence Lys762-Arg763-Ser764, identical to endothelial cell-associated proteolysis. This conclusion stems from the observation that substitution of Arg763 by a glycine residue completely abolishes proteolytic processing. As a result, transfection of COS-1 with the mutant vWF-Gly763 cDNA does not significantly affect the multimeric organization of secreted vWF molecules. Consequently, we conclude that proteolytic processing of pro-vWF is not required for multimer formation. Pulse-chase labeling of COS-1 cells transfected with full-length vWF cDNA reveals pro-vWF exclusively in cell lysates, whereas both pro-vWF and mature vWF are encountered in the conditioned medium. These observations indicate that proteolytic processing of pro-vWF is a "late" event during intracellular routing of these molecules or may occur extracellularly.     

人ZP3基因的RT-PCR cDNA克隆

目的：研究人ZP3基因的结构及构建人ZP3基因原核表达系统。方法：从人卵巢组织中分离出mRNA并以此作为模版,通过RT—PCR扩增出人ZP3基因cDNA片段,然后将其克隆在pUC18质粒上,并对克隆片段进行序列分析。结果：共克隆到ZP3-A（1300bp）、ZP3-B（1180bp）、ZP3-C（1200bp）和ZP3-D（1080bp）4种不同长度的人ZP3基因cDNA片段,对其中最长的ZP3-A片段的测序结果表明,它包含了人ZP3基因阅读框内的全部序列,与NCBI Sequence Viewer中公布的人ZP3 mRNA序列（NM-007155）相比较,在1275bp长的编码区内只有一个碱基不同,两者同源性达到99．92％。结论：本研究克隆到的ZP3-A cDNA片段确是人ZP3基因无疑。     

Identification of bacterial clones encoding bovine caseins by direct immunological screening of the cDNA library

A sensitive immunoassay was used to identify recombinant plasmids carrying cDNA fragments of bovine caseins in the cDNA library from bovine mammary gland mRNA. Colonies grown on nitrocellulose filters were lysed in situ and proteins from the lysates were blotted onto CNBr-activated cellulose filter paper. Antigens covalently bound to CNBr-activated paper or bound to nitrocellulose filters were detected by reaction with antiserum to caseins, followed by ¹²⁵I-labelled Staphylococcus aureus protein A and autoradiography. Six clones were found positive among 5400 of the cDNA library: 3-A1, 3-B2, 3-B5, 3-H7, 2-A5 and 2-C9. The molecular weights of chimeric pre-β-lactamase: casein proteins synthesized in Escherichia coli were estimated by immunoblotting. Colony hybridization and nucleotide sequence analysis showed that clone 3-B5 contained a cDNA fragment of bovine χ-casein, clone 3-H7 contained a cDNA fragment of β-casein, while clones 2-A5 and 2-C9 carried cDNA fragments of α_si-casein.     

The variable C-terminal extension of G-protein-coupled receptor kinase 6 constitutes an accessorial autoregulatory domain

G-protein-coupled receptor kinases (GRK) are known to phosphorylate agonist-occupied G-protein-coupled receptors. We expressed and functionally characterized mouse GRK6 proteins encoded by four distinct mRNAs generated by alternative RNA splicing from a single gene, mGRK6-A to mGRK6-D. Three isoforms, mGRK6-A to mGRK6-C differ in their C-terminal-most portion, which is known to mediate membrane and/or receptor interaction and regulate the activity of GRK4-like kinases. One isoform, mGRK6-D, is identical to the other mGRK6 variants in the N-terminal region, but carries an incomplete catalytical domain. Mouse GRK6-D was catalytically inactive and specifically present in the nucleus of transfected cells. Recombinant mouse GRK6-A to mGRK6-C were found to be membrane-associated in cell-free systems and in transfected COS-7 cells, suggesting that the very C-terminus of GRK6-A, lacking in GRK6-B and mGRK6-C and carrying consensus sites for palmitoylation, is not required for membrane interaction. Interestingly, the shortest catalytically active variant, mGRK6-C, was conspicuously more active in phosphorylating light-activated rhodopsin than mGRK6-A and mGRK6-B, implying that the C-terminus of the latter two variants may fulfil an autoinhibitory function. Mutation and removal of C-terminal-most region of mGRK6-A by site-directed mutagenesis revealed that this region contains three autoregulatory elements: two discontinuous inhibitory elements consisting of a single residue, D560, and the sequence between residues S566 and L576, and an intervening stimulatory element. The results suggest that mGRK6-C may be considered a basic, prototypic representative of the GRK4-like kinases, which is capable of interacting with both plasma membrane and its receptor substrate, but is resistant to further regulatory modification conferred to the prototype via C-terminal extension.     

Characterization of recombinant von Willebrand factor corresponding to mutations in type IIA and type IIB von Willebrand disease.

Type IIA and IIB von Willebrand disease (vWD) result from defects in von Willebrand factor (vWF). Although both type IIA and IIB vWD are characterized by the absence of high molecular weight multimers in plasma, vWF from patients with type IIA vWD demonstrates a decreased affinity for the platelet receptor glycoprotein Ib (GPIb), whereas vWF from patients with type IIB vWD show an increased affinity for GPIb. To investigate how structural alterations in vWF affect its interaction with GPIb, we reproduced the reported potential mutations in type IIA and IIB vWD in vWF cDNA and expressed the recombinant proteins in COS-1 cells. The type IIA vWF potential mutation was represented by a G-->A transversion which results in the substitution of Lys for Glu at position 875 in the mature vWF subunit (rvWFLys875). The type IIB vWF mutation corresponds to a duplicated ATG codon, resulting in three contiguous methionines starting at position 540-541 in the normal vWF sequence (rv-WFduplMet540-541). The subunit composition and multimeric structure of both mutant proteins were similar to the wild-type rvWF. The rvWFLys875 bound to fixed platelets in the presence of ristocetin similar to wild-type rvWF. The rvWFduplMet540-541 bound to fixed platelets in the absence of agonist. The rvWFLys875 appears to interact normally with GPIb, and the decreased affinity for the platelet receptor observed in plasma is probably a consequence of prior reduction in multimeric size resulting from the defect. In contrast, the duplication of Met540-541 increases the reactivity of vWF for its platelet receptor.     

cDNA sequences for human von Willebrand factor reveal five types of repeated domains and five possible protein sequence polymorphisms

A human umbilical vein endothelial cell cDNA library in lambda gt11 was screened with two previously described cDNA inserts for human von Willebrand factor. Among 16 positive isolates, two that hybridized with a probe corresponding to the amino terminus of von Willebrand factor were sequenced. Together, these four cDNA inserts span 6.5 kilobases of the von Willebrand factor mRNA sequence, completely specifying the 2050 amino acids of the subunit of mature, secreted von Willebrand factor and 24 residues of a precursor peptide. Approximately 77% of the sequence is contained in five types of repeated domains. Domain A consists of 193-220 amino acids and is present in three tandem copies between residues 497 and 1111. Domain B contains 25-35 amino acids and is present in three copies between residues 1533 and 1636. Domain C consists of 116-119 amino acids and is duplicated between residues 1637 and 1899. In contrast to the essentially contiguous repetition of domains A-C, the two copies of domains D and E are each separated by 804 and 1383 amino acids, respectively. Domain D1 contains 289 amino acids between residues 79 and 367, while domain D2 consists of 270 amino acids between residues 1171 and 1440. Domain E1 consists of 46 amino acids between residues 25 and 70, and domain E2 consists of 46 amino acids between residues 1453 and 1498. The triplicated A domains are notably poor in Cys content, while the remaining domains are Cys-rich. The A domains appear to be homologous to a 225-residue segment of complement factor B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning and sequencing of a cDNA coding for mature human kidney cathepsin H

A cDNA library was established from human kidney RNA and screened with an extended oligonucleotide probe derived from the amino-acid sequence of human cathepsin H. A recombinant clone, pRF15, was isolated and characterized. DNA sequence analysis of its 1106-nucleotide-long insert revealed that pRF15 encodes the complete protein sequence of mature cathepsin H plus 28 amino acids of a propeptide, thus confirming that cathepsin H is synthesized as a larger precursor molecule and posttranslationally processed. Northern blot analysis indicated that cathepsin H is predominantly synthesized in kidney. A high degree of sequence homology was observed with rat cathepsin H, especially within the propeptide. The part of the prosequence coding for the "minichain" is conserved in the prosequence of aleurain, a plant thiol protease.     

小麦tae-MIR156前体基因的克隆及其靶基因TaSPL17多态性分析

Squamosa-promoter binding protein (SBP)-box基因是植物特有的一类转录因子, 广泛参与植物生长发育, 其部分成员受miR156调控。文章克隆了小麦(Triticum aestivum) tae-MIR156前体基因, 转录后能够形成茎环结构。小麦10个SBP-box基因中, 仅TaSPL3和TaSPL17在编码区存在tae-miR156识别位点。SPL17在普通小麦的A基因组供体种乌拉尔图小麦(Triticum urartu, AA) UR209和B基因组供体种拟斯卑尔脱山羊草(Aegilops speltoides, BB) Y2001中均为多拷贝(SPL17-A1、SPL17-A2和SPL17-A3; SPL17-B1、SPL17-B2和SPL17-B3), 在D基因组供体种粗山羊草(Aegilops tauschii, DD) Ae38中仅检测到一种序列(SPL17-D); SPL17-A2与SPL17-B2, SPL17-A3与SPL17-B3、SPL17-D两两之间序列的一致性程度均大于99%, 且与普通小麦(中国春、衡观35和双丰收)的TaSPL17序列具有较高的一致性, 提示它们可能来源于共同的祖先基因, 并且在进化过程中高度保守。靶基因TaSPL17中的tae-miR156识别位点非常保守, 在根据单株穗数和基因型多样性挑选的SubP1和SubP2群体中均未检测到tae-miR156识别位点存在变异碱基。     

Analysis of DNA from human adenovirus type 6 with restriction endonucleases HindIII, BglIII and BamHI

The genome of the type 6 human adenovirus has three restriction sites for R.BamHI, thirteen for R.HindIII and ten for R.BglII. The terminal fragments of DNA cleaved by each of the enzymes have been determined by means of terminal nucleotidyl transferase and by analysis of the DNA-terminal protein complex. The sequence of the cleaved fragments has been determined by partial cleavage of DNA, simultaneous digestion of DNA with various combinations of enzymes and secondary digestion of individual isolated fragments with other enzymes. The following order of the cleaved fragments in the adenovirus type 6 genome has been found (the figures in brackets are the weights in mega-daltons): for R.BamHI-B(7.1)-D(3.0)-C(4.05)-A(8.5); for R.HindIII-F(1.7)-C1(2.14)-A(3.44)-M(0.046)-I(1.24)-J(0.77)-D(2.1)-E(1.96)-B(3.18)-H(1.36)-L(0.18)-C2(2.14)-G(1.44)-K(0.16); for R.BglII-E(2.07)-B(3.58)-A(4.8)-C(3.36)-I(0.78)-D(3.25)-G(1.37)-J(0.21)-F(1.85)-K(0.17)-H(0.94).     

Structure of an antifreeze polypeptide and its precursor from the ocean pout, Macrozoarces americanus

Serum antifreeze polypeptides (AFP) from Newfoundland ocean pout have been resolved by ion exchange chromatography and reverse phase high performance liquid chromatography into at least 12 components. The protein sequences of three of the AFP were determined using a combination of protein Edman degradation and cDNA sequencing. The AFP precursor protein encodes for a preprotein of 87 amino acids with no obvious prosequences. Two of the AFP (SP1-A and SP1-C) were separate gene products with minor amino acid sequence differences. The protein structure of SP1-C precursor is MKSVILTGLLFVLLCVDHMTASQSVVAT QLIPINTALTPAMMEGKVTNPIGIPFAEMSQIVGKQVNTPVAKGQTLMPNMVKTYVAGK. The third AFP (SP1-B) is a post-translation modification product of SP1-C. These experiments indicate that the ocean pout AFP are a multigene family with protein structure different from any other known polypeptide antifreezes.     

p190-A, a human tumor suppressor gene, maps to the chromosomal region 19q13.3 that is reportedly deleted in some gliomas

To date, two distinct genes coding for Ras GAP-binding phosphoproteins of 190kDa, p190-A and p190-B, have been cloned from mammalian cells. Rat p190-A of 1513 amino acids shares 50% sequence identity with human p190-B of 1499 amino acids. We have previously demonstrated, using rat p190-A cDNA, that full-length p190-A is a tumor suppressor, reversing v-Ha-Ras-induced malignancy of NIH 3T3 cells through both the N-terminal GTPase (residues 1-251) and the C-terminal Rho GAP (residues 1168-1441) domains. Here we report the cloning of the full-length human p190-A cDNA and its first exon covering more than 80% of this protein, as well as its chromosomal mapping. Human p190-A encodes a protein of 1514 amino acids, and shares overall 97% sequence identity with rat p190-A. Like the p190-B exon, the first exon of p190-A is extremely large (3.7 kb in length), encoding both the GTPase and middle domains (residues 1-1228), but not the remaining GAP domain, suggesting a high conservation of genomic structure between two p190 genes. Using a well characterized monochromosome somatic cell hybrid panel, fluorescent in situ hybridization (FISH) and other complementary approaches, we have mapped the p190-A gene between the markers D19S241E and STD (500 kb region) of human chromosome 19q13.3. Interestingly, this chromosomal region is known to be rearranged in a variety of human solid tumors including pancreatic carcinomas and gliomas. Moreover, at least 40% glioblastoma/astrocytoma cases with breakpoints in this region were previously reported to show loss of the chromosomal region encompassing p190-A, suggesting the possibility that loss or mutations of this gene might be in part responsible for the development of these tumors.     

RNA binding specificity of a Drosophila snRNP protein that shares sequence homology with mammalian U1-A and U2-B" proteins.

We have characterized a recombinant Drosophila melanogaster RNA binding protein, D25, by virtue of its antigenic relationship to mammalian U1 and U2 small nuclear ribonucleoprotein (U snRNP) proteins. Sequence analysis revealed that D25 bears strong similarity to both the human U1 snRNP-A (U1-A) and U2 snRNP-B" (U2-B") proteins. However, at residues known to be critical for the RNA binding specificities of U1-A and U2-B" D25 sequence is more similar to U2-B". Using direct RNA binding assays D25 selected U1 RNA from either HeLa or Drosophila Kc cell total RNA. Furthermore, D25 bound U1 RNA when transfected into mammalian cells. Thus, D25 appears to be a Drosophila homolog of the mammalian U1-A protein, despite its sequence similarity to U2-B".