Expression and characterisation of the thrombospondin type I repeats of human properdin

Properdin, an upregulator of the alternative complement pathway, is central to deposition of the activated complement fragment C3b on the surfaces of the pathogens, which it achieves by preventing the dissociation of the Bb catalytic subunit from the inherently labile C3bBb complexes. It is also known to bind sulphated glycoconjugates, such as sulphatides. Properdin has an unusual structure formed by oligomerisation of a rod-like monomer into cyclic dimers, trimers and tetramers. The monomer (approximately 53 kDa) contains an N-terminal region of no known homology, followed by six non-identical repeats of 60 amino acids (based on exon/intron boundaries), called 'thrombospondin type I repeats' or TSR modules. We have expressed and purified the N-terminal region and each of the individual TSR repeats in Escherichia coli. Although the individual recombinant TSRs, after a denaturation-renaturation cycle, appeared to be correctly folded modules, as judged by the one-dimensional (1D)- and 2D-nuclear magnetic resonance spectra of TSR3, they did not show binding to either C3b or sulphatide. Polyclonal antibodies were raised against each TSR and were found to be module-specific. The anti-TSR5 polyclonal antibody was found to inhibit binding of native human properdin to solid-phase C3b, or sulphatides. It could also block properdin-dependent haemolysis of rabbit erythrocytes. These results are consistent with the view that the TSR5 contains the major site in properdin which is involved in both C3b and sulphatide binding. It also suggests that a co-operative intramolecular interaction between TSRs, as found in the native molecule, is required for TSR5 to bind either C3b or sulphatides.     

Expression and mutagenesis of thrombospondin.

Thrombospondin is a 420,000-dalton adhesive glycoprotein that is composed of three subunits of equivalent molecular weight. When the cDNA for the complete coding region of the human endothelial cell thrombospondin subunit is expressed in mouse NIH 3T3 cells, a 420,000-dalton protein is synthesized and secreted. The expressed protein comigrates with human platelet thrombospondin both in the presence and in the absence of a reducing agent. The expressed protein binds to a monoclonal anti-thrombospondin antibody, heparin, and calcium. In addition to the 420,000-dalton protein, the transfected cell lines also express a variable amount of a 140,000-dalton polypeptide. When the culture supernatants that are produced by cells that are expressing thrombospondin are applied to heparin-Sepharose, the 420,000-dalton and the 140,000-dalton proteins are bound to the column and are eluted with buffer containing 0.55 and 0.3 M NaCl, respectively. The 140,000-dalton protein only binds to heparin-Sepharose in the presence of calcium. Deletion of the region of homology with procollagen results in defective assembly of the trimer. Deletion of the type 1 or type 2 repeats results in decreased stability of the subunit with the predominant polypeptides that are expressed having molecular weights of 127,000 and 130,000, respectively. These polypeptides retain low-affinity heparin-binding activity. High-affinity heparin binding is markedly diminished by mutations in either of two sequence motifs that include clusters of lysines and arginines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression and initial characterization of a recombinant human thrombospondin heparin binding domain

Thrombospondin (TSP) is a trimeric glycoprotein of Mr 420,000. It was originally described as a major component of human platelet alpha granules and is essential for the secondary phase of platelet aggregation. TSP is also synthesized and secreted by a variety of nucleated cells where it functions in processes involving growth and adhesion of cells to the extracellular matrix. Many of these processes are heparin-inhibitable and are mediated by a proteolytic fragment of TSP called the heparin binding domain (HBD). In order to facilitate the analysis of the structure and function(s) of this domain, we have expressed this molecule in Escherichia coli. A fragment of a TSP cDNA that encodes the heparin binding domain was inserted into the prokaryotic expression vector pJBL6. In bacterial cells grown at 42 degrees C, this vector directs the synthesis of a 24,000-Da polypeptide. Milligram quantities of this protein were purified to homogeneity from E. coli lysates. The structure of the recombinant HBD was confirmed by protein sequencing. The protein was further characterized by analysis of its conformation and function. The recombinant HBD binds [3H]heparin with a Kd of 71 nM, almost identical to that of TSP-derived HBD (80 nM). Additionally, the recombinant HBD is able to compete for TSP binding to 11B carcinoma cells. These studies indicate that the recombinant HBD is synthesized and purified in a native configuration and is functionally equivalent to thrombospondin-derived HBD. They further indicate that glycosylation of the thrombospondin HBD is not necessary for its interaction with heparin and that sequences essential to this interaction reside within the first 229 amino acids of secreted thrombospondin.     

A unique proteolytic fragment of human fibrinogen containing the A alpha COOH-terminal domain of the native molecule

The COOH-terminal portion of the A alpha chain of human fibrinogen is highly susceptible to proteolytic degradation. This property has prevented isolation of the COOH-terminal domain of fibrinogen for the direct investigation of its functional characteristics. Human fibrinogen was degraded with hementin, a fibrinogen-olytic protease from the posterior salivary glands of the leech, Haementeria ghilianii. Two initial fragments, Yhem1 and Dhem1, produced by cleavage through the three polypeptide chains in the connector region, were characterized and shown to retain the entire A alpha COOH-terminal domain. Late cleavages by hementin occurred in the A alpha chain COOH-terminal region to produce fragments Yhem and Dhem with shorter A alpha chain remnants. Fragments Dhem were isolated from an intermediate hementin digest of fibrinogen using anion-exchange chromatography. Fragment Dhem1 was separated further from Dhem fragments with shorter alpha chain remnants by affinity chromatography on immobilized plasma fibronectin. Fragment Dhem1 represents a unique proteolytic fragment of fibrinogen containing an intact A alpha chain COOH-terminal region. NH2-terminal sequence analysis of isolated chains from fragment Dhem1 located hementin cleavage sites in the connector region to A alpha Asn102-Asn103, B beta Lys130-Gln131, and gamma Pro76-Asn77. The specific interaction of fragment Dhem1 with immobilized fibronectin indicated that the binding site probably was located within the COOH-terminal 111 amino acids of the A alpha chain. The overall pattern of fibrinogen cleavage by hementin is similar to that of plasmin, yet hementin cleaves preferably in the coiled-coil connector, sparing the A alpha COOH-terminal domain.     

Expression of members of the thrombospondin family by human skeletal tissues and cultured cells.

We have previously shown that the multifunctional platelet glycoprotein thrombospondin-1 (TSP-1) promotes resorption in an in vitro resorption assay. However, TSP-1 is one of a family of multifunctional TSP molecules, and the current study was undertaken to investigate whether it is TSP-1 or another TSP family member which may be involved in regulation of resorption in vivo. RT-PCR was performed on cultured human bone cells, cultured human chondrocytes, and three separate samples of human osteoclastoma tissue using primers specific for each TSP family member. mRNA for TSP-2 was detected in almost all samples, and significantly in all osteoclastomas in the above tissues, while TSP-1 was detected less frequently and was only seen in one of three osteoclastomas. TSP-3, -4, and COMP were detected only in a minority of cases. These results indicate that TSP-2 is the most common TSP family member found in skeletal tissues and that TSP-2, rather than TSP-1, may be the molecule responsible for promoting resorption in vivo.     

Expression of cell adhesion molecule T-cadherin in the human vasculature

Alterations in expression of surface adhesion molecules on resident vascular and blood-derived cells play a fundamental role in the pathogenesis of cardiovascular disease. Smooth muscle cells (SMCs) have been shown to express T-cadherin (T-cad), an unusual GPI-anchored member of the cadherin family of adhesion molecules. Particular relevance for T-cad in cardiovascular tissues is indicated by our present screen (immunoblotting) of human tissues and organs whereby highest expression of T-cad was found in aorta, carotid, iliac and renal arteries and heart. To explore the (patho)physiological role for T-cad in the vasculature we performed an immunohistochemical analysis of T-cad expression in normal human aorta and atherosclerotic lesions of varying severity. T-cad was present both in the intima and media and was expressed in endothelial cells (ECs), SMCs and pericytes, but not in monocytes/macrophages, foam cells and lymphocytes. In the adventitia T-cad was present in the wall of vasa vasorum and was expressed in ECs, SMCs and pericytes. T-cad was differentially expressed in SMCs from distinct vascular layers of normal aorta (for example, high in the subendothelial (proteoglycan) layer of the intima, low in the musculoelastic intimal layer and in the media), as well as at different stages of lesion progression. In SMCs there was an apparent inverse relationship between the intensities of T-cad and smooth muscle alpha-actin expression, this being most prominent in lesions. The findings suggest a phenotype-associated expression of T-cad which may be relevant to control of the normal vascular architecture and its remodelling during atherogenesis.     

Structure and chromosomal localization of the human thrombospondin gene

Thrombospondin (THBS1) is a large modular glycoprotein component of the extracellular matrix and contains a variety of distinct domains, including three repeating subunits (types I, II, and III) that share homology to an assortment of other proteins. Determination of THBS1 gene structure has revealed that the type I repeat modules are encoded by symmetrical exons and that the heparin-binding domain is encoded by a single exon. To further elucidate the higher level organization of THBS1, the gene was localized to the q11-qter region of chromosome 15.     

Relative abundance of thrombospondin 2 and thrombospondin 3 mRNAs in human tissues.

The levels of thrombospondin 2 (TSP2) and thrombospondin 3 (TSP3) mRNAs in a variety of human tissues were determined by analysis of multiple-tissue mRNA dot blots. For TSP2 mRNA, aorta and fetal heart had the greatest relative abundance. High levels were also detected for muscle, fetal, endocrine, immune, and nerve tissues. The pattern of expression of TSP3 mRNA was very different: kidney, pituitary gland, trachea, uterus, and fetal kidney had the greatest abundance. In general, TSP3 mRNA was expressed at high levels in endocrine, muscle, and fetal tissues. In addition to the tissue-specific differences, a more even distribution of TSP3 mRNA among tissues was observed. The high relative abundance of the two mRNAs in a variety of tissues and the tissue-specific differences in expression could be significant for understanding the diverse roles implicated for TSP2 and TSP3.     

The structure of human platelet thrombospondin

Two distinct murine monoclonal antibodies, designated MA-I and MA-II, and limited proteolysis with thrombin and trypsin have been used to probe the structure of human platelet thrombospondin. The results indicate that each of the constituent chains of thrombospondin comprise four distinct polypeptide segments. The production of these segments is influenced by the presence of calcium, the enzyme employed, the temperature of digestion, and the enzyme-to-substrate ratio. Thrombin digestion in the presence of calcium results in the release of a 30,000-dalton fragment, designated segment I, which contains the epitope for MA-II and the heparin-binding site. Prior EDTA treatment results in the concomitant cleavage of a 25,000-dalton fragment, designated segment IV, from the other terminus. Limited tryptic digestion in the absence of calcium produces a 47,000-dalton fragment (segment III) which is adjacent to segment IV. Segment III contains the epitope for MA-I. Segment II is an 85,000-dalton fragment which contains the interchain disulfide bonds. Calcium inhibits proteolysis at cleavage sites between segments II and III and between segments III and IV. In the presence of calcium, an 85,000-dalton fragment is produced, which is derived from portions of segments II, III, and possibly IV. Electron microscopy of platinum replicas produced by low angle rotary shadowing reveals that thrombospondin is composed of four well-defined globular regions connected by thin flexible regions. Three of the globular regions, designated globular region C, appear to be at the ends of the three thin connecting regions. The fourth globular region, designated globular region N, appears to be close to the site where the chains are cross-linked. Globular region N can be resolved into three separate smaller globular structures which are 70 +/- 7.1 A in diameter. This region is selectively removed by thrombin digestion in the presence of calcium and binds a monoclonal antibody directed against the heparin-binding peptides. These data indicate that globular region N comprises the three NH2-terminal portions (segment I) from each of the three chains of thrombospondin. Globular region C is located at the ends of each of the three thin connecting regions which are each approximately 291 +/- 46 A long. The removal of calcium results in a decrease in the size of globular region C from 118 +/- 18.6 A to 80 +/- 7.4 A and an increase in the length of the adjacent thin connecting region to 383 +/- 30 A.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular analysis redefines three human chromosome 14 deletions

We have used a panel of 13 DNA markers in the distal region of chromosome 14q to characterize deletions in three patients determined cytogenetically to have a ring or terminally deleted chromosome 14. We have characterized one patient with a ring chromosome 14 [r (14) (p13q32.33)] and two with terminal deletions [del (14) (pterq32.3:)]. The two patients with cytogenetically identical terminal deletions of chromosome 14 were found to differ markedly when characterized with molecular markers. In one patient, none of the markers tested were deleted, indicating that the apparent terminal deletion is actually due to either an undetected interstitial deletion or a cryptic translocation event. In the other patient, the deletion was consistent with the cytogenetic observations. The deleted chromosome was shown to be of paternal origin. The long-arm breakpoint of the ring chromosome was mapped to within a 350-kb region of the immunoglobulin heavy chain gene cluster (IGH). This breakpoint was used to localize markers D14S20 and D14S23, previously thought to lie distal to IGH, to a more proximal location. The ring chromosome represents the smallest region of distal monosomy 14q yet reported.     

Internal deletions in human interleukin-6: structure-function analysis

By cDNA mutagenesis, we have constructed internal and C-terminal deletions (delta 21-51, delta 52-97, delta 97-104, delta 127-174, delta 97-184 and delta 134-184) in human interleukin-6 (hIL-6). All those deletion-carrying hIL-6 (delta hIL-6) proteins were then produced in Xenopus laevis oocytes and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results show that, at least in frog oocytes, the first potential N-glycosylation site (Asn45) is utilized exclusively. The IL-6 conformation of these deletion-carrying proteins has been studied by immunoprecipitation with two kinds of monoclonal antibodies (mAb's): mAb's that show preference towards denatured hIL-6, or conformation-specific mAb's. The binding pattern of these two series of mAb's indicated that the IL-6 conformation has been largely destroyed for four of our delta-proteins. Proteins delta 21-51 and delta 127-174 have kept a part of the IL-6 tertiary structure since they are still recognized by some conformation-specific mAb's. All of these delta hIL-6 proteins were inactive in the IL-6 hybridoma growth factor (HGF) assay and unable to inhibit the HGF activity of the recombinant human wild-type IL-6 (wt hIL-6). Moreover, the oocyte-synthesized delta hIL-6 (delta 21-51, delta 127-174, delta 97-184, delta 134-184) did not bind to the IL-6 receptor. Finally, we have produced two proteins with aa 29-33 or 97-104 substituted by corresponding murine IL-6 (mIL-6) sequences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Members of the thrombospondin gene family bind stromal interaction molecule 1 and regulate calcium channel activity

The thrombospondins (TSPs) are a family of matricellular proteins that regulate cellular phenotype through interactions with a myriad of other proteins and proteoglycans. We have identified a novel interaction of the members of the TSP gene family with stromal interaction molecule 1 (STIM1). This association is robust since it is preserved in Triton X-100, can be detected with multiple anti-TSP-1 and anti-STIM1 antibodies, and is detected in a wide range of cell types. We have also found that STIM1 co-immunoprecipitates with TSP-4 and cartilage oligomeric matrix protein (COMP), and that a recombinant version of the N-terminal domain of STIM1 binds to the signature domain of TSP-1 and COMP. The association of the TSPs with STIM1 is observed in both the presence and absence of calcium indicating that the calcium-dependent conformation of the signature domain of TSPs is not required for binding. Thus, this interaction could occur in the ER under conditions of normal or low calcium concentration. Furthermore, we observed that the expression of COMP in HEK 293 cells decreases STIM1-mediated calcium release activated calcium (CRAC) channel currents and increases arachidonic acid calcium (ARC) channel currents. These data indicate that the TSPs regulate STIM1 function and participate in the reciprocal regulation of two channels that mediate calcium entry into the cell.     

Biosynthesis and secretion of thrombospondin in human melanoma cells

A polyclonal antisera specific for human platelet thrombospondin (TSP) has been utilized to study the biosynthesis and secretion of TSP in the M21 human melanoma cell line. Pulse-chase indirect immunoprecipitation analysis reveal that human melanoma cells rapidly synthesize and secrete this platelet alpha-granule associated glycoprotein. Topographical analysis of the melanoma cell surface by indirect immunofluorescence indicate that the TSP molecules have no obvious extracellular organization. The implications of thrombospondin synthesis in the metastatic process of melanoma are discussed.     

Neutron and X-ray scattering studies on the human complement protein properdin provide an analysis of the thrombospondin repeat

Properdin is a regulatory glycoprotein of the alternative pathway of the complement system of immune defense. It is responsible for the stabilization of the C3 convertase complex formed between C3b and the Bb fragment of factor B. Neutron and X-ray solution scattering experiments were performed on the dimeric and trimeric forms of properdin. These have RG values of 9.1 and 10.7 nm, respectively. The scattering curves were compared with Debye sphere modeling simulations for properdin. Good agreements were obtained for models similar to published electron micrographs showing that the properdin trimer has a triangular structure with sides of 26 nm. Such a structure also accounted for sedimentation coefficient data on properdin. Primary structure analyses for mouse and human properdin have shown that this contains six homologous motifs known as the thrombospondin repeat (TSR), which is the second most abundant domain type found in the complement proteins. Sequences for these 12 TSRs were aligned with 19 others found in thrombospondin and the late complement components. Three distinct groups of TSRs were identified, namely, the TSRs found in thrombospondin and properdin, the TSRs mostly found at the N-terminus of the late complement components, and the TSRs found at the C-terminus of the late components. Averaged secondary structure predictions suggested that all three groups contain similar backbone structures with two amphipathic turn regions and one hydrophilic beta-strand region. The mean dimensions of the TSRs of properdin in solution were determined to be approximately 4 nm X 1.7 nm X 1.7 nm, showing that these are elongated in structure.     

Complex formation of human thrombospondin with osteonectin

Human thrombospondin, a 450-kDa glycoprotein isolated from platelets and endothelial cells, specifically interacts with osteonectin, a protein of 30 kDa isolated from bovine bones and human platelets. Using ELISA, purified osteonectin binds to solid-phase-adsorbed thrombospondin with a dissociation constant (Kd) of 0.7 nM. Binding of thrombospondin to solid-phase-adsorbed osteonectin was also observed (Kd = 0.86 nM). The interaction of thrombospondin with solid-phase-adsorbed osteonectin was significantly decreased (81% inhibition) when using an excess of fluid-phase osteonectin. Thrombospondin-osteonectin complex formation was calcium-dependent as shown by a 50-80% inhibition in the presence of EDTA. None of the proteins known to interact with thrombospondin (fibrinogen, fibronectin, collagen, plasminogen) had a significant inhibitory effect on thrombospondin-osteonectin complex formation. This selective interaction was confirmed by affinity chromatography. Iodinated osteonectin, previously incubated with purified thrombospondin, specifically bound to an anti-thrombospondin monoclonal antibody (P10) linked to protein-A--Sepharose 4B. Elution of the anti-thrombospondin antibody from protein A allowed the recovery of the thrombospondin-osteonectin complex in the eluate, as judged by SDS/polyacrylamide gel electrophoresis and autoradiography. Blotting of purified thrombospondin to osteonectin adsorbed onto nitrocellulose further confirmed complex formation. In addition, when released from thrombin-stimulated platelets, thrombospondin and osteonectin bound to anti-thrombospondin IgG-coated plates indicating that osteonectin was complexed to thrombospondin once the platelet-release reaction has occurred.     

Protein engineering and properties of human metalloproteinase and thrombospondin 1

This work generated many truncated proteins and Glu(385) to Ala (E(385)/A) mutants of the human metalloproteinase and thrombospondin 1 (METH-1 or ADAMTS1) and specific antibodies. METH-1 was an active endopeptidase and both the metalloproteinase and the disintegrin/cysteine-rich domains were required for the proteinase activity. A point mutation at the zinc-binding site (E(385)/A) abolished the catalytic activity. METH-1 protein function may be modulated through proteolytic cleavage at multiple sites. One 135 kDa species had an NH(2)-terminal sequence of L(33)GRPSEEDEE. A species at 115 kDa and some other protein bands began with F(236)VSSHRYV(243), indicating that METH-1 proenzyme might be activated by a proprotein convertase such as furin by cleaving the R(235)-F(236) peptide bond. This cleavage was not an autocatalytic process since the E(385)/A mutants were also processed. Furthermore, a 52 kDa band with an NH(2)-terminal sequence of L(800)KEPLTIQV resulted from the digestion between the first and the second thrombospondin 1-like motifs in the spacer region of the extracellular matrix-binding domains.     

Interaction of thrombospondin with resting and stimulated human platelets

The interaction of isolated and radioiodinated thrombospondin with washed human platelets has been characterized. The ligand bound to nonstimulated and thrombin-stimulated platelets in a time-dependent manner, and apparent steady state was reached within 25 min. Binding was not due to iodination of the ligand and was inhibited by nonlabeled thrombospondin but not by unrelated proteins, and bound ligand was identical with thrombospondin in terms of subunit structure. Nonlinear curve-fitting analyses of binding to resting platelets suggested the presence of a single class of sites which bound 3,100 +/- 1,000 molecules/platelet with an apparent Kd of 50 +/- 20 nM. This interaction was not attributable to contaminating cells or inadvertant platelet activation. Binding to thrombin-stimulated platelets had a lower apparent affinity (Kd = 250 +/- 100 nM) and higher apparent capacity (35,600 +/- 9,600 molecules/platelet). Thrombin-enhanced binding was dependent upon agonist dose and platelet stimulation. Fibrinogen, a monoclonal antibody to GPIIb-IIIa, temperature, and divalent ions had differential effects upon thrombospondin binding to resting and stimulated platelets, suggesting the presence of two distinct mechanisms of thrombospondin binding to platelets. While thrombospondin binding to thrombin-stimulated platelets occurs with characteristics similar to those observed for fibrinogen, fibronectin, and von Willebrand Factor, its high affinity interaction with resting platelets is unique to this adhesive glycoprotein.     

Light microscopic immunolocation of thrombospondin in human tissues

Affinity-purified antisera against thrombospondin were used to locate the presence of this glycoprotein in frozen sections of several human tissues by immunofluorescence techniques. Immunostaining was observed in the peritubular connective tissue and in basement membrane regions beneath glandular epithelium in skin and lung. Intense immunostaining was observed at the dermal-epidermal junction in skin and in small blood vessels throughout this tissue. Skeletal muscle exhibited positive staining with anti-thrombospondin antisera within interstitial areas. Immunostaining was confined to the luminal portions of large blood vessels such as aorta. In large blood vessels that contained lesions of atherosclerosis, immunostaining was observed throughout the lesion area and was especially prominent surrounding some of the lesion cells. These results indicate that thrombospondin is located within the matrix of a variety of human tissues and supports the suggestion that this glycoprotein is an endogenous component of some extracellular matrices.     

Structural study of the sugar chains of human platelet thrombospondin

The asparagine-linked sugar chains of human platelet thrombospondin were released as oligosaccharides by hydrazinolysis. About 12 mol of sugar chains was released from one thrombospondin molecule. This was converted to radioactive oligosaccharides by sodium borotritide reduction after N-acetylation, and separated into one neutral and four acidic fractions by paper electrophoresis. More than 90% of the oligosaccharides were recovered in the acidic fraction. The acidic oligosaccharides were mostly converted to neutral oligosaccharides by sialidase treatment, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were further fractionated by Bio-Gel P-4 column chromatography. Structural study of each oligosaccharide by sequential exoglycosidase digestion and methylation analysis revealed that the thrombospondin contains mono-, bi-, tri-, and tetraantennary complex-type sugar chains in addition to a small amount of high-mannose type. Approximately 70% of the complex-type sugar chains was fucosylated at asparagine-linked N-acetylglucosamine residue and 19% of the biantennary complex-type sugar chains was bisected.