Expression of spasmolysin (FIM-A.1): an integumentary mucin from Xenopus laevis

In the past, a unique type of precursor for a secretory protein was discovered. It contains a central repetitive domain rich in threonine residues and terminal cysteine-rich domains. Due to striking homologies of these terminal domains with pancreatic spasmolytic polypeptide, originally the name "prepro-spasmolysin" was proposed. Here we show that the mature protein has a MW of about 130 kDa, consisting of about 70% carbohydrate and 30% protein. Similar O-linked glycoproteins have been found in mucins from human intestine. For this and numerous other reasons we decided to rename this glycoprotein "frog integumentary mucin A.1" (FIM-A.1). Furthermore, analysis of the protein with specific antibodies against the predicted C-terminal end indicates that FIM-A.1 is probably not processed at pairs of basic residues. In situ hybridization as well as immunofluorescence studies revealed that FIM-A.1 is expressed and stored exclusively in mature mucous glands of Xenopus laevis skin. Only cone cells at the proximal part of these glands do not synthesize FIM-A.1. In contrast, all other physiologically active peptides from X. laevis skin investigated so far are synthesized in granular glands. A hypothetical function of FIMs for defense against microbial infections is discussed.     

The role of Willebrand factor in platelet - blood vessel interaction, including a discussion of resistance to atherosclerosis in pigs with von Willebrand's disease

Von Willebrand pigs have all the manifestations of the severe human disease. The role of Willebrand antigen (VIII R:AG) and ristocetin cofactor (VIII: RWF) was assessed in these pigs by (1) transfusion and (2) "in vitro" bleeding time assay. The skin bleeding time became normal when the level of transfused Willebrand factor (VIII R:AG/RWF) was raised in the plasma above 30 U/dl. After single or repeated transfusions, skin capillary endothelium and platelets were still distinguished from normal by VIII R:AG deficiency. When incisions in excised porcine skin ("in vitro" bleeding time) were perfused with blood and plasma fractions, haemostasis occurred when plasmatic Willebrand factor exceeded 30 U/dl whether the skin or platelets came from normal or from von Willebrand pigs. The platelet plug occluding the skin incision contained VIII R:AG by immunofluorescence. Willebrand factor appears to coat surfaces and to serve as a platelet attachment protein. These bleeder pigs are resistant to atherosclerosis. If platelets are involved in early atherosclerotic lesions, the role of Willebrand factor in platelet - blood vessel interaction may be important.     

Binding of ADAMTS13 to von Willebrand factor

ADAMTS13, a metalloprotease, cleaves von Willebrand factor (VWF) in plasma to generate smaller, less thrombogenic fragments. The interaction of von Willebrand factor with specific ADAMTS13 domains was characterized with a binding assay employing von Willebrand factor immobilized on a plastic surface. ADAMTS13 binding was saturable and reversible. Equilibrium binding occurred within 2 h and the half-time for dissociation was approximately 4 h. Binding to von Willebrand factor was similar with either recombinant ADAMTS13 or normal plasma ADAMTS13; plasma from a patient who lacked ADAMTS13 activity showed no binding. The stoichiometry of binding was one ADAMTS13 per two von Willebrand factor monomers, and the K(d) was 14 nm. The ADAMTS13 metalloprotease and disintegrin domains did not bind VWF detectably. ADAMTS13 truncated after the first thrombospondin type 1 repeat bound VWF with a K(d) of 206 nm, whereas ADAMTS13 truncated after the spacer domain had a K(d) of 23 nm, which is comparable with that of full-length ADAMTS13. Truncation after the eighth thrombospondin type 1 repeat reduced the binding affinity by approximately 3-fold and truncation after the seventh thrombospondin type 1 repeat in addition to the CUB domains increased the affinity for von Willebrand factor by approximately 2-fold. Therefore, the spacer domain is required for ADAMTS13 binding to von Willebrand factor. The first thrombospondin repeat also affects binding, and the C-terminal thrombospondin type 1 and CUB domains of ADAMTS13 may modulate this interaction.     

Cleavage of von Willebrand factor requires the spacer domain of the metalloprotease ADAMTS13

ADAMTS13 consists of a reprolysin-type metalloprotease domain followed by a disintegrin domain, a thrombospondin type 1 motif (TSP1), Cys-rich and spacer domains, seven more TSP1 motifs, and two CUB domains. ADAMTS13 limits platelet accumulation in microvascular thrombi by cleaving the Tyr1605-Met1606 bond in von Willebrand factor, and ADAMTS13 deficiency causes a lethal syndrome, thrombotic thrombocytopenic purpura. ADAMTS13 domains required for substrate recognition were localized by the characterization of recombinant deletion mutants. Constructs with C-terminal His6 and V5 epitopes were expressed by transient transfection of COS-7 cells or in a baculovirus system. No association with extracellular matrix or cell surface was detected for any ADAMTS13 variant by immunofluorescence microscopy or chemical modification. Both plasma and recombinant full-length ADAMTS13 cleaved von Willebrand factor subunits into two fragments of 176 kDa and 140 kDa. Recombinant ADAMTS13 was divalent metal ion-dependent and was inhibited by IgG from a patient with idiopathic thrombotic thrombocytopenic purpura. ADAMTS13 that was truncated after the metalloprotease domain, the disintegrin domain, the first TSP1 repeat, or the Cys-rich domain was not able to cleave von Willebrand factor, whereas addition of the spacer region restored protease activity. Therefore, the spacer region is necessary for normal ADAMTS13 activity toward von Willebrand factor, and the more C-terminal TSP1 and CUB domains are dispensable in vitro.     

von Willebrand factor is a cofactor for thrombin-catalyzed cleavage of the factor VIII light chain

The proteolytic activation of highly purified, heterodimeric porcine factor VIII and factor VIII-von Willebrand factor complex by thrombin was compared at I 0.17, pH 7.0, 22 degrees C. During the activation of factor VIII, heavy-chain cleavage is necessary to activate the procoagulant function, whereas light-chain cleavage is required to dissociate factor VIII from von Willebrand factor. The kinetics of activation of free factor VIII and factor VIII-von Willebrand factor complex were identical. The steady-state kinetics of thrombin-catalyzed heavy-chain cleavages and light-chain cleavage of factor VIII either free or in complex with von Willebrand factor were studied using sodium dodecyl sulfate-polyacrylamide gel radioelectrophoresis and scanning densitometry of fragments derived from 125I-labeled factor VIII. Association of factor VIII with von Willebrand factor resulted in an 8-fold increase in the catalytic efficiency (kcat/Km) of light-chain cleavage (from 7 x 10(6) to 54 x 10(6) M-1 s-1). The catalytic efficiencies of heavy-chain cleavage at position 372 (approximately 6 x 10(6) M-1 s-1) and position 740 (approximately 100 x 10(6) M-1 s-1) were not affected by von Willebrand factor. We conclude that von Willebrand factor promotes cleavage of the factor VIII light chain by thrombin which is followed by rapid dissociation of the complex, so that the rate-limiting step becomes heavy-chain cleavage at position 372. This accounts for the observation that von Willebrand factor has no effect on the kinetics of activation of factor VIII by thrombin.     

N-terminal cleavage of the salivary MUC5B mucin. Analogy with the Van Willebrand propolypeptide?

Sequence similarities between the oligomeric mucins (MUC2, MUC5AC, MUC5B) and the von Willebrand factor suggest that they may be assembled in a similar way. After oligomerization, a fragment corresponding to the D1 and D2 domains is released from the von Willebrand factor. This cleavage does not appear to occur in pig submaxillary mucin, the only mammalian mucin in which this cleavage has been examined thus far, but whether other oligomeric mucins undergo N terminus proteolysis is not known. Antibodies recognizing the D1, D2, D3, and the first Cys domains in MUC5B were established and used to investigate to what extent proteolytic cleavage occurs within the N-terminal part of salivary MUC5B. The antibodies against the D1 and D2 domains identified a polypeptide corresponding in size to a MUC5B fragment generated by cleavage within the D' domain analogously with the von Willebrand factor propolypeptide. The antibodies did not recognize the main mucin population, suggesting that the major part of salivary MUC5B is subjected to this cleavage. An antibody recognizing the D3 domain was used to reveal a second cleavage site in the "soluble" but not in the "insoluble" MUC5B fraction: the first structural difference observed between soluble and insoluble salivary MUC5B. The identification of these cleavage events shows that the N-terminal sites for MUC5B oligomerization are present in the D3 domain and/or in domains located C-terminal to this part of the molecule.     

Perturbation of cultured human vascular endothelial cells by phorbol ester or thrombin alters the cellular von Willebrand factor distribution

We have studied the influence of perturbation of cultured human umbilical vein endothelial cells on the distribution of the von Willebrand factor. As shown previously, short-term (less than 1 hr) treatment of endothelial cells with the phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) or thrombin resulted in the release of cellular stored von Willebrand factor. Long-term treatment with PMA or thrombin evoked a distinct change in the endothelial cell distribution of von Willebrand factor, evident 24 to 48 hrs after exposure. Whereas the contents of the von Willebrand factor storage sites in the cells were gradually restored within 48 hrs, enhanced amounts of von Willebrand factor were secreted into the medium. However, PMA did not increase the endothelial cell contents of mRNA encoding for von Willebrand factor. The number as well as the size of von Willebrand factor storage granules in the endothelial cells increased after exposure to the phorbol ester, as determined by immunofluorescence microscopy. A second treatment with PMA or thrombin, 48 hrs after cells had been stimulated with these agents, resulted again in the instantaneous release of von Willebrand factor. PMA and thrombin caused a decrease in the von Willebrand factor contents of the extracellular matrix. Pulse-chase experiments revealed that PMA blocked the deposition of von Willebrand factor in the subendothelium, whereas PMA did not affect the degradation of matrix von Willebrand factor. Thus, perturbation of endothelial cells changes the cellular distribution of von Willebrand factor.     

Endothelial cell-derived high molecular weight von Willebrand factor is converted into the plasma multimer pattern by granulocyte proteases

We have previously found that the von Willebrand factor released by cultured human umbilical vein endothelial cells appeared as a single high molecular weight band in glyoxyl agarose electrophoresis. In the present studies we report that this high molecular weight endothelial cell-derived von Willebrand factor, when incubated with granulocyte lysates, was cleaved into a series of multimers indistinguishable from those seen in normal plasma (or type II von Willebrand disease). This von Willebrand factor-cleaving activity was released from granulocytes by calcium ionophore A23187 but was not detected in cytosolic fractions depleted of granular contents. It was inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride. This von Willebrand factor-cleaving activity thus provides a possible mechanism for the generation of plasma von Willebrand factor multimers from the high molecular weight form of von Willebrand factor secreted by endothelial cells.     

Crystal structure of the A domain from complement factor B reveals an integrin-like open conformation

Complement factor B is a 90 kDa protein consisting of three domains: a three-module complement control protein, a von Willebrand factor A domain, and a C-terminal serine protease (SP) domain that adopts a default inactive (zymogen) conformation. The interaction between factor B and pathogen-bound C3b is mediated by its A domain, triggering a conformational change in factor B that ultimately creates the "C3 convertase" of the alternative complement pathway. We report the crystal structure of the A domain from factor B and show that it contains an integrin-like MIDAS motif that adopts the "open" conformation typical of integrin-ligand complexes, with an acidic residue (provided by a fortuitous crystal contact) completing the coordination of the metal ion. Modeling studies indicate that the factor B A domain can also adopt the closed conformation, supporting the hypothesis that an "integrin-like switch" is conserved in complement proteins and perhaps in 60 other A domains found within the human proteome.     

Exposure of platelet binding sites in von Willebrand factor by adsorption onto polystyrene latex particles

Von Willebrand factor molecules are flexible linear polymers composed of repeating protomeric polypeptide subunits. In the process of primary hemostasis, von Willebrand factor promotes platelet adhesion and platelet plug formation at the site of vascular injury. This biologic activity is apparently related to the multimeric size of von Willebrand factor. We simulated von Willebrand factor binding to the subendothelial surface by adsorbing purified human von Willebrand factor onto polystyrene latex particles of two different diameters, i.e., 0.312 μm and 2.02 μm. The rate and extent of ¹²⁵I-labeled von Willebrand factor binding to polystyrene was similar with both size classes of latex particles. The von Willebrand factor-coated latex beads of 2.02 μm diameter, in contrast to the smaller size, induced rapid agglutination of formalin-fixed human platelets in the absence of any other aggregating agent. Von Willebrand factor was also adsorbed from human plasma onto latex particles coated with anti-von Willebrand factor antibodies. Again, only the large beads, carrying the von Willebrand factor-antibody complex, induced agglutination of fixed platelets. Shear stress promoted the rate of von Willebrand factor adsorption to latex particles. Our results suggest that adsorption to surface exposes binding sites in human von Willebrand factor for platelets.     

The A-domain and the thrombospondin-related motif of Plasmodium falciparum TRAP are implicated in the invasion process of mosquito salivary glands.

Sporozoites from all Plasmodium species analysed so far express the thrombospondin-related adhesive protein (TRAP), which contains two distinct adhesive domains. These domains share sequence and structural homology with von Willebrand factor type A-domain and the type I repeat of human thrombospondin (TSP). Increasing experimental evidence indicates that the adhesive domains bind to vertebrate host ligands and that TRAP is involved, through an as yet unknown mechanism, in the process of sporozoite motility and invasion of both mosquito salivary gland and host hepatocytes. We have generated transgenic P.berghei parasites in which the endogenous TRAP gene has been replaced by either P.falciparum TRAP (PfTRAP) or mutated versions of PfTRAP carrying amino acid substitutions or deletions in the adhesive domains. Plasmodium berghei sporozoites carrying the PfTRAP gene develop normally, are motile, invade mosquito salivary glands and infect the vertebrate host. A substitution in a conserved residue of the A-domain or a deletion in the TSP motif of PfTRAP impairs the sporozoites' ability to invade mosquito salivary glands. Notably, midgut sporozoites from these transgenic parasites are still able to infect mice. Midgut sporozoites carrying a mutation in the A-domain of PfTRAP are motile, while no gliding motility could be detected in sporozoites with a TSP motif deletion.     

cDNA for the carboxyl-terminal region of a rat intestinal mucin-like peptide.

When subjected to thiol reduction, purified intestinal mucins have been shown to undergo a decrease in molecular mass and to liberate a 118-kDa glycopeptide (Roberton, A. M., Mantle, M., Fahim, R. E. F., Specian, R., Bennick, A., Kawagishi, S., Sherman, P., and Forstner, J. F. (1989) Biochem. J. 261, 637-647). The latter has been called a putative "link" component because it is assumed to be important for disulfide bond-mediated mucin polymerization. Controversy exists as to whether the putative link is an integral mucin component or a separate mucin-associated glycopeptide. In the present study both NH2-terminal and internal amino acid sequences of the 118-kDa glycopeptide of rat intestinal mucin were used to generate opposing oligonucleotide primers for polymerase chain reaction. A specific 1.2-kilobase (kb) product was obtained, from which a 0.5-kb HindIII fragment was used as a probe to screen a lambda ZAP II cDNA library of rat intestine. A 2.6-kb cDNA (designated MLP 2677) was sequenced and revealed an open reading frame of 2.5 kb encoding 837 amino acids. The deduced amino acid sequence showed that the putative link peptide is equivalent to the carboxyl-terminal 689 amino acids of a larger peptide. Northern blots revealed a mRNA size of approximately 9 kb. Computer searches revealed no sequence homology with other proteins, but similarities were seen in the alignment of cysteine residues in the link and in several domains of human von Willebrand factor, as well as cysteine-rich areas of bovine and porcine submaxillary mucins and a frog skin mucin designated FIM-B.1. In keeping with earlier demonstrations of the presence of mannose in the 118-kDa glycopeptide, there were several (13) consensus sequences for attachment of N-linked oligosaccharides within the link domain. Further sequencing of MLP 2677 in a direction 5' to the codon specifying the NH2-terminal proline of the link has revealed a coding region for 148 amino acids, including a unique 75-amino acid domain rich in cysteine and proline, and a region containing 4.5-variable tandem repeats (each 11-12 amino acids) rich in serine, threonine, and proline. The presence of mucin-like tandem repeats suggests that the entire cysteine-rich link peptide represents the carboxyl-terminal region (75.5 kDa) of a mucin-like peptide (MLP). The latter is estimated to have a molecular mass of approximately 300 kDa.     

Solution structure and dynamics of a prototypical chordin-like cysteine-rich repeat (von Willebrand Factor type C module) from collagen IIA

Chordin-like cysteine-rich (CR) repeats (also referred to as von Willebrand factor type C (VWC) modules) have been identified in approximately 200 extracellular matrix proteins. These repeats, named on the basis of amino acid conservation of 10 cysteine residues, have been shown to bind members of the transforming growth factor-beta (TGF-beta) superfamily and are proposed to regulate growth factor signaling. Here we describe the intramolecular disulfide bonding, solution structure, and dynamics of a prototypical chordin-like CR repeat from procollagen IIA (CR(ColIIA)), which has been previously shown to bind TGF-beta1 and bone morphogenetic protein-2. The CR(ColIIA) structure manifests a two sub-domain architecture tethered by a flexible linkage. Initial structures were calculated using RosettaNMR, a de novo prediction method, and final structure calculations were performed using CANDID within CYANA. The N-terminal region contains mainly beta-sheet and the C-terminal region is more irregular with the fold constrained by disulfide bonds. Mobility between the N- and C-terminal sub-domains on a fast timescale was confirmed using NMR relaxation measurements. We speculate that the mobility between the two sub-domains may decrease upon ligand binding. Structure and sequence comparisons have revealed an evolutionary relationship between the N-terminal sub-domain of the CR module and the fibronectin type 1 domain, suggesting that these domains share a common ancestry. Based on the previously reported mapping of fibronectin binding sites for vascular endothelial growth factor to regions containing fibronectin type 1 domains, we discuss the possibility that this structural homology might also have functional relevance.     

Characterization and mapping of the 5′ portion of von Willebrand factor pseudogene

A genomic fragment containing the 5 boundary of the von Willebrand factor pseudogene was cloned, partially sequenced and used for in situ hybridization experiments on metaphase spreads from a Philadelphia chromosome (Ph¹)-positive chronic myelogenous leukemia patient. Data obtained indicate that the von Willebrand factor pseudogenic region is centromeric to the breakpoint cluster region on 22q11.2. This probe could be used for the study of deletions in the DiGeorge syndrome.     

Primary structure of the leukocyte function-associated molecule-1 alpha subunit: an integrin with an embedded domain defining a protein superfamily

The leukocyte function-associated molecule 1 (LFA-1, CD11a/CD18) is a membrane glycoprotein which functions in cell-cell adhesion by heterophilic interaction with intercellular adhesion molecule 1 (ICAM-1). LFA-1 consists of an alpha subunit (Mr = 180,000) and a beta subunit (Mr = 95,000). We report the molecular biology and protein sequence of the alpha subunit. Overlapping cDNAs containing 5,139 nucleotides were isolated using an oligonucleotide specified by tryptic peptide sequence. The mRNA of 5.5 kb is expressed in lymphoid and myeloid cells but not in a bladder carcinoma cell line. The protein has a 1,063-amino acid extracellular domain, a 29-amino acid transmembrane region, and a 53-amino acid cytoplasmic tail. The extracellular domain contains seven repeats. Repeats V-VII are in tandem and contain putative divalent cation binding sites. LFA-1 has significant homology to the members of the integrin superfamily, having 36% identity with the Mac-1 and p150,95 alpha subunits and 28% identity with other integrin alpha subunits. An insertion of approximately 200 amino acids is present in the NH2-terminal region of LFA-1. This "inserted/interactive" or I domain is also present in the p150,95 and Mac-1 alpha subunits but is absent from other integrin alpha subunits sequenced to date. The I domain has striking homology to three repeats in human von Willebrand factor, two repeats in chicken cartilage matrix protein, and a region of complement factor B. These structural features indicate a bipartite evolution from the integrin family and from an I domain family. These features may also correspond to relevant functional domains.     

Isolation and characterization of a collagen binding domain in human von Willebrand factor

von Willebrand factor binds to fibrillar type I collagen in a rapid, temperature-independent, reversible, specific, and saturable manner. Evaluation of binding isotherms by Scatchard-type analysis demonstrated that 6-18 micrograms of von Willebrand factor bind per mg of collagen, with Ka between 2 and 8 X 10(8) M-1. Five distinct tryptic fragments, purified under denaturing and reducing conditions and representing over 75% of the molecular mass of the von Willebrand factor subunit, were tested for their capacity to inhibit the von Willebrand factor-collagen interaction. Complete inhibition was obtained with a 52/48-kDa fragment at a concentration of approximately 1 microM. The location of this fragment in the subunit was established to be between Val-449 and Lys-728. Fifteen monoclonal antibodies against the 52/48-kDa fragment inhibited von Willebrand factor binding to collagen. Six antibodies against other portions of the von Willebrand factor subunit had no inhibitory effect. The tryptic fragment was a competitive inhibitor of von Willebrand factor binding to collagen and, therefore, recognizes the same interaction site as the intact molecule. These studies precisely define a domain in the von Willebrand factor subunit that interacts with type I collagen.     

Aberrant expression of histo-blood group A type 3 antigens in vascular endothelial cells in inflammatory sites.

Histo-blood group ABH antigens are widely distributed in human tissues. The epitopes of ABH antigens are carried by at least four different peripheral core isotypes of internal carbohydrate backbones (type 1-4). Each type of ABH antigen is expressed tissue specifically, and aberrant expression of ABH antigens is often observed during oncogenesis. We immunohistochemically examined the expression of A type 3 antigens in wounded and diseased skin tissues (A and AB blood groups). In uninjured skin, the expression of A type 3 antigens was restricted to the eccrine sweat gland. In addition to the sweat glands, A type 3 antigens were found in vascular endothelial cells of the wound sites. The extent of A type 3 antigens expression related to postinfliction intervals. A significantly higher expression rate of A type 3 antigens in endothelial cells was also observed in diseased skin, suggesting that inflammation might induce A type 3 antigen expression in endothelial cells. Double-color immunofluorescence staining of the specimens showed that von Willebrand factor (vWF) was a core-protein of A type 3 determinants aberrantly expressed in endothelial cells in inflamed tissues, suggesting that aberrant expression of A type 3 antigens is involved in stabilization of vWF in inflammation.