Production and purification of recombinant human SPARC.

The matricellular protein SPARC (secreted protein acidic and rich in cysteine, also known as osteonectin or as BM-40) is a collagen-binding protein with a capacity to induce cell rounding and influence proliferation in cultured cells. In mice that do not express SPARC, fibrillar collagen is reduced in some adult tissues; notably, a reduction in fibrosis is reported in response to fibrotic stimuli in lungs, heart, skin, liver, and in the eye. Recently, mutations in the gene encoding SPARC were found in patients afflicted with osteogenesis imperfecta. Thus, SPARC appears to be a critical mediator of collagen deposition and assembly in tissues. A useful tool for assessing the function of SPARC in ECM assembly is a source of purified recombinant SPARC. Outlined in this chapter is a brief discussion of different strategies for generating recombinant SPARC and an experimental strategy for producing and purifying human recombinant SPARC driven by baculoviral expression in insect cells.

Osteonectin/SPARC is a product of articular chondrocytes/cartilage and is regulated by cytokines and growth factors.

Rabbit articular chondrocytes maintained in monolayer, synthesized and secreted a 46 kDa protein into the culture medium. N-terminal sequence analysis and immunoprecipitation of the radiolabeled material revealed this protein to be osteonectin (ON)/SPARC, a protein previously shown to be present in bone. When chondrocytes were exposed to interleukin-1, a cytokine with matrix degradative properties, ON synthesis and secretion was greatly inhibited. However, this was specific to IL-1 since two other pro-inflammatory cytokines (tumor-necrosis factor-alpha and interleukin-6) with properties similar to IL-1, failed to cause any discernible effect on ON synthesis. Several growth factors (TGF-beta, PDGF, and IGF-1), that have been shown to stimulate other cartilage matrix macromolecular synthesis, also stimulated ON synthesis and were also able to reverse the inhibitory effect of IL-1 on ON synthesis. These observations were also demonstrated in explant cultures of cartilage. Our studies suggest that ON is a biosynthetic product of articular cartilage and could play a role in cartilage structure and/or function.

SPARC from corneal epithelial cells modulates collagen contraction by keratocytes.

PURPOSE: Contraction of the scar tissue during corneal wound healing changes the shape of the cornea and corneal refraction. In a previous study, it was found that corneal epithelial cells secrete the factor that stimulates collagen gel contraction by keratocytes in vitro. The purpose of the present study was to purify and identify the contraction-stimulating factor derived from corneal epithelial cells. METHODS: The cultured medium of rabbit corneal epithelial cells was collected and used as an epithelial cell-conditioned medium (ECCM). Subcultured rabbit keratocytes were embedded in a collagen gel, and collagen gel contraction was investigated. The contraction-stimulating factor in the ECCM was purified through acetone precipitation, affinity chromatography (heparin Sepharose), gel filtration, and reversed-phase chromatography. The amino acid sequence of a contraction-stimulating factor was analyzed. RESULTS: Collagen gel contraction by keratocytes was enhanced by the addition of ECCM in a dose-dependent manner. The amino acids sequence of the contraction-stimulating factor was homologous to a 32-kDa glycoprotein, a secreted protein that is acidic and rich in cysteine (SPARC). Western blot analysis confirmed that SPARC was contained in the ECCM. Collagen gel contraction by keratocytes was enhanced by the addition of purified SPARC in a dose-dependent manner. SPARC was found in the basal layer of the migrating epithelium and activated keratocytes adjacent to the wound 3 days and 1 week after perforating injury in rabbit corneas. CONCLUSIONS: Epithelial cells secrete SPARC, which modulates the contraction of scar tissue in the corneal stroma.

Proliferation, differentiation and characterization of osteoblasts from human BM mesenchymal cells.

BACKGROUND: The objective of this study was to isolate osteoprogenitor cells (OPC) from BM mesenchymal stromal cells (MSC) and test their capacity to proliferate and differentiate into osteoblasts. METHODS: Human MSC were separated on a Percoll gradient and cultured in DMEM supplemented with 15% human serum, and characterized by flow cytometric analyzes for CD34, CD13, CD90, CD105 and CD117. To induce differentiation, cultured cells were exposed to 10(-7) m dexamethasone (dexa) and/or 10(-3) m sodium beta-glycerophosphate (beta-GlyP) and 1,25-dihydroxyvitamin D3 (calcitriol) or 9-cis-retinoic acid (9-RA). RESULTS: alkaline phosphatase (AP) activity was detected in cells irrespective of the dexa and/or beta-GlyP treatment. Antigenic phenotypes of MSC were CD34- (more than 99%) and CD13+ CD90+ CD105+ CD117+ (c. 50%). The treatment induced extracellular calcium deposition and gene and protein expression of osteonectin (ON) and bone sialoprotein (BSP): beta-GlyP induced an increase (c. 2.2-fold) of the ON gene and dexa augmented (c. 2.7-fold) the gene expression of BSP II. Gene expression of BSP I reached a maximum at 3 weeks of combined treatment. Osteocalcin gene expression was induced only after additional treatment with calcitriol or 9-RA. Ultrastructural analysis revealed the secretory phenotype of OPC. DISCUSSION: Under appropriate treatment, MSC can give rise to OPC that have the capacity to differentiate into osteoblasts characterized by the expression of osteogenic markers, osteoblastic properties and stromal BM cells phenotypes. These cells may represent a promising material to be utilized in orthopedic cellular therapy.

Porcine SPARC: isolation from dentin, cDNA sequence, and computer model.

Genes encoding the major enamel matrix proteins and non-collagenous proteins of bone and dentin are members of the secretory calcium-binding phosphoprotein (SCPP) family, which originated from ancestral SPARC (secreted protein, acidic and rich in cysteine; BM-40/osteonectin). To better understand the role of SPARC in mineralizing systems, we isolated SPARC from developing pig teeth, deduced its primary structure from the cDNA sequence, and determined its quaternary structure by homology modelling with reference to human SPARC crystal structures. The guanidine/EDTA extract from porcine dentin was fractionated by anion-exchange and size-exclusion chromatography. Stains-all positive bands at 38 and 35 kDa gave the N-terminal sequences APQQEALPDETEV and DFEKNYNMYIFPV, which corresponded to the SPARC N terminus and an internal region of the protein. Porcine SPARC contains 300 amino acids, including the 17-amino acid signal peptide, and shares 96.2% amino acid sequence identity with human SPARC. Without post-translational modifications, the 283-amino acid secreted protein has a molecular mass of 32.3 kDa. The three-dimensional model revealed that porcine SPARC contains a single N-linked glycosylation at N113, seven intramolecular disulfide bridges, and assembles into dimers. SPARC is composed of three structural/functional domains: an acidic Ca2+-binding, a follistatin-like, and an extracellular calcium-binding domain.

Secreted modular calcium-binding protein-1 localization during mouse embryogenesis.

BM-40 is an extracellular matrix-associated protein and is characterized by an extracellular calcium-binding domain as well as a follistatin-like domain. Secreted modular calcium-binding protein-1 (SMOC-1) is a new member of the BM-40 family. It consists of two thyroglobulin-like domains, a follistatin-like domain and a new domain without known homologues and is expressed ubiquitously in many adult murine tissues. Immunofluorescence studies, as well as immunogold electron microscopy, have confirmed the localization of SMOC-1 in or around basement membranes of adult murine skin, blood vessels, brain, kidney, skeletal muscle, and the zona pellucida surrounding the oocyte. In the present work, light microscopic immunohistochemistry has revealed that SMOC-1 is localized in the early mouse embryo day 7 throughout the entire endodermal basement membrane zone of the embryo proper. SMOC-1 mRNA is synthesized, even in early stages of mouse development, by mesenchymal as well as epithelial cells deriving from all three germ layers. In embryonic stage day 12, and fetal stages day 14, 16, and 18, the protein is present in the basement membrane zones of brain, blood vessels, skin, skeletal muscle, lung, heart, liver, pancreas, intestine, and kidney. This broad and organ-specific distribution suggests multifunctional roles of SMOC-1 during mouse embryogenesis.

Purification of a calcium binding protein (rat SPARC) from primary Sertoli cell-enriched culture medium.

A protein with an apparent Mr of 43,000 was purified from Sertoli cell-enriched culture medium by sequential anion-exchange, gel permeation, C4 reversed-phase, and diphenyl reversed-phase HPLC. N-Terminal sequence analysis of this protein revealed a sequence of NH2-XPQTEAAEEMVAEETVV for the first 17 amino acids. Comparison of this sequence with existing protein data base maintained at the Protein Identification Resource revealed that it shares extensive identity with a previously described protein secreted by mouse embryo parietal endoderm, SPARC, which is equivalent to a protein secreted by a basement-membrane-producing tumor, BM-40; and a bone protein, osteonectin. This protein also possesses similar in vitro biological activity of SPARC in which it binds Ca2+. The possible physiological significance of this protein was discussed.

Characterization of mineral-binding 40-kDa glycoprotein extracted from young adult rabbit alveolar bone.

A forty-kilodalton (40-kDa) protein was extracted from alveolar bone of young adult rabbit with 0.5 M EDTA after extraction with 4 M GuHCl, and purified by gel-filtration, anion-exchange and hydroxyapatite columns using a high-pressure liquid chromatography system under denaturing conditions. The purified 40-kDa protein was not susceptible to bacterial collagenase and thrombin, but was cleaved by cyanogen bromide. The protein was stained blue with Stains-all. Among various lectins, concanavalin A and lentil lectin agglutinin bound to this protein, but peanut agglutinin, Ricinus communis agglutinin, phytohemagglutinin-E and wheatgerm lectin agglutinin did not. Lectin binding assays showed that the protein is a glycoprotein containing large amounts of mannose and/or glucose residues, but is not a fragment of proteoglycan. The amino acid composition of the protein shows a characteristically high content of acidic amino acids. Therefore, the mineral-binding 40-kDa glycoprotein is considered to be osteonectin/secreted protein acidic and rich in cysteine (SPARC), in terms of similarities to bovine and porcine osteonectins with regard to molecular weight and contents of glycoses and amino acids.

Characterization of human osteoblast and megakaryocyte-derived osteonectin (SPARC).

Osteonectin is an adhesive, cell, and extracellular matrix-binding glycoprotein found primarily in the matrix of bone and in blood platelets in vivo. Osteonectins isolated from these two sources differ with respect to the complexity of their constituent N-linked oligosaccharide. In this study, osteonectin synthesized by bone-forming cells (osteoblasts) and platelet-producing cells (megakaryocytes) in vitro was analyzed to determine if the proteins produced were analogous in terms of glycosylation to those isolated from bone and platelets, respectively. Immunoblot analyses of osteonectin produced by the osteoblast-like cell lines, SaOS-2 and MG-63, indicated that secreted and intracellular forms of the molecule are structurally distinct. Endoglycosidase treatment and immunoblotting of osteonectin secreted from SaOS-2 and MG-63 cells, under serum-deprived conditions, suggested that the molecule possessed a complex type oligosaccharide unlike the high-mannose moiety found on bone matrix-derived osteonectin. Biosynthetic labeling of SaOS-2 cells and human megakaryocytes indicated that both cell types synthesize osteonectin de novo. Electrophoretic and glycosidase sensitivity analyses of [35S]-osteonectin isolated from lysates of metabolically labeled SaOS-2 cells and megakaryocytes indicated that these two cell types synthesize osteonectin molecules that are identical in oligosaccharide structure to the isolated bone and platelet proteins. These data suggest that the intracellular form of the osteonectin molecule is glycosylated differently in SaOS-2 cells and megakaryocytes but that the extracellular form which is secreted from platelets in vivo and osteoblasts in vitro is characterized by the presence of a complex type N-linked oligosaccharide.

Demonstration of osteonectin mRNA in megakaryocytes: the use of the polymerase chain reaction.

Platelets have been shown to release osteonectin on thrombin stimulation. The origin of platelet osteonectin was unclear as it may have been synthesized by megakaryocytes or it may have been endocytosed from plasma as other platelet alpha-granule constituents are. Platelet osteonectin has a larger apparent molecular size than the bone species, although the molecular basis for this difference has not been elucidated. These two issues have been addressed here by (1) examining the potential for osteonectin biosynthesis in human megakaryocytes by demonstrating the presence of osteonectin mRNA in purified megakaryocytes, and (2) comparing the coding portion of osteonectin transcript in megakaryocytes to the size of its bone counterpart. Because of the limitations of cell population purity and in obtaining sufficient numbers of megakaryocyte cells for Northern analysis, we have used the polymerase chain reaction (PCR) to detect the presence of human osteonectin mRNA in megakaryocyte and megakaryocyte-depleted bone marrow cells. Isolation of RNA, cDNA synthesis, and PCR were performed on human osteosarcoma SaOS-2 cells, enriched megakaryocytes, and megakaryocyte-depleted cells. Restriction enzyme analysis of PCR DNA products confirmed the identity of the products as those encoding osteonectin for all three cell populations studied. In addition, the sizes of DNA indicate that osteonectin genomic DNA, nuclear RNA, or altered transcript were not amplified, and that the transcript from megakaryocytes is the same size as that from bone cells. These data suggest that the difference in protein size between platelet and bone osteonectin is due to posttranslational modification. To overcome the possibility that megakaryocyte signal originated from contaminating cells (less than 5% by cell count), all three cell populations were diluted to less than one cell per tube and PCR amplification was performed. Limiting dilution analyses demonstrated the presence of osteonectin mRNA in single megakaryocytes as well as in single cells from the cell population depleted of megakaryocytes, suggesting the capacity for osteonectin biosynthesis in all cells studied. The procedure we describe in this report can be used to examine specific characteristics of mRNA molecules in heterogeneous cell populations and in situations where only small quantities of cells can be obtained.

Conformational changes of bovine bone osteonectin induced by interaction with calcium.

To clarify calcium-induced conformational changes in bovine bone osteonectin, the protein was labeled with fluorescein isothiocyanate (FITC) in the presence and absence of calcium. By calcium titration using fluorescence spectrometry, it was demonstrated that FITC-osteonectin labeled in the presence of 2 mM CaCl2 showed a much higher affinity for calcium ions than did that labeled in the absence of calcium ions. The midpoint for completion of the increase in the intrinsic fluorescence (K0.5) of the two were 1 x 10(-7) M and 5 x 10(-7) M, respectively. By tryptic digestion and isolation of the fluorescent peptide of both FITC-osteonectins, the site of FITC-labeling was determined to be Lys174. Furthermore, it was found that the efficacy of labeling in this specific binding site was three times higher in the FITC-osteonectin labeled in the presence of 2 mM CaCl2 than in that labeled in the absence of calcium. The results indicate that in the presence of 2 mM CaCl2 the microenvironment around Lys174 of osteonectin was more open to modification than in the absence of calcium.

Purification of SPARC/osteonectin.

SPARC is a matricellular protein that regulates cell adhesion, extracelluolar matrix production, growth factor activity and cell cycle. This unit describes the purification of SPARC, also termed osteonectin and BM/40, from cultured mammalian cells. Additional information is presented on the purification of recombinant SPARC (rSPARC) from E. coli and from Sf9 cells, as well as its isolation from blood platelets. Assays for the activity of SPARC, de-adhesion and inhibition of cellular proliferation in vitro, are described. The expression of SPARC during remodeling and repair tissue in response to injury identifies it as a therapeutic target for the treatment of fibrotic disease, certain cancers and other disorders in which regulation of angiogenesis is a key factor.

Specific interaction of SPARC with endothelial cells is mediated through a carboxyl-terminal sequence containing a calcium-binding EF hand.

SPARC is a secreted, Ca(2+)-binding protein that modulates cell shape and gene expression (Sage, E.H., and Bornstein, P. (1991) J. Biol. Chem. 266, 14831-14834). In the present study we questioned whether SPARC interacted with an endothelial cell surface receptor. The binding of 125I-SPARC to bovine aortic endothelial cells was dependent on Ca2+ and was sensitive to small changes in extracellular pH; maximal binding occurred at pH 7.1. Scatchard analysis indicated approximately 2.3 x 10(7) binding sites/cell with an apparent KI of 1.1 nM. The interaction was diminished specifically by competition with synthetic peptides corresponding to amino acids 54-73 (SPARC 54-73) and 254-273 (SPARC254-273). The binding of 125I-SPARC254-273, a sequence containing a Ca(2+)-binding EF-hand, was saturated within 45 min at a concentration of 5 microM; Scatchard analysis indicated 4.2 x 10(7) sites/cell and a KI of 2.4 nM. Iodinated proteins from plasma membranes were affinity-chromatographed on SPARC254-273; several proteins with apparent masses ranging from 153 to 100 kDa (unreduced) or from 153 to 122 kDa (reduced) were eluted with the soluble peptide. These proteins represent candidates for a SPARC receptor(s) that mediates the biological activity of this protein on endothelial cells.

Biosynthesis of bone proteins [SPP-1 (secreted phosphoprotein-1, osteopontin), BSP (bone sialoprotein) and SPARC (osteonectin)] in association with mineralized-tissue formation by fetal-rat calvarial cells in culture.

To determine the relationship between the expression of bone proteins and the formation of mineralized-tissue matrix, the biosynthesis of non-collagenous bone proteins was studied in cultures of fetal-rat calvarial cells, which form mineralized nodules of bone-like tissue in the presence of beta-glycerophosphate. The temporal pattern of protein synthesis in both mineralizing and non-mineralizing cultures was studied by metabolic labelling with [35S]methionine, 35SO4(2-) or 32PO4(3-) over a 5-day period. After a 24 h labelling period, the culture media were harvested and the cell layers extracted sequentially with aq. 0.5 M-NH3, followed by 4 M-guanidinium chloride (GdmCl), 0.5 M-EDTA and a second extraction with 4 M-GdmCl. Protein associated with collagenous bone matrix was analysed after digestion with bacterial collagenase. On the basis of [35S]methionine labelling, the major proteins extracted from the mineralizing matrix were secreted phosphoprotein-1 (SPP-1; osteopontin), bone sialoprotein (BSP) and a 14 kDa phosphoprotein. The presence of SPP-1 and BSP in the conditioned media of both mineralizing and non-mineralizing cultures and their incorporation into the mineralizing nodules indicated that these proteins associate with preformed mineral crystals. However, some BSP was also present in GdmCl extracts and, together with a 35 kDa sulphated protein, was released from a bacterial-collagenase digestion of the tissue residue in both non-mineralizing and mineralizing cultures. Two forms of sulphated SPP-1 were identified, a highly phosphorylated 44 kDa species being the predominant form in the mineralized matrix. The BSP was more highly sulphated but less phosphorylated than SPP-1. Bone SPARC (secreted protein, acid and rich in cysteine) protein (osteonectin) was present almost entirely in the conditioned media and did not incorporate 32PO4(3-) or 35SO4(2-). The SPP-1 and the 14 kDa protein were susceptible to thrombin digestion, the 44 kDa SPP-1 being specifically cleaved into 28 and 26 kDa fragments. The fragments were labelled uniformly with [35S]methionine, but the 28 kDa fragment incorporated more 35SO4(2-), but less 32PO4(3-), than the 26 kDa fragment. These studies demonstrate that SPP-1 and BSP are the major osteoblast-derived bone proteins to bind to the bone mineral. That BSP also binds to the collagenous bone matrix indicates a potential role for this protein in linking the hydroxyapatite with collagen.

Heat-shock response in cultured chick embryo chondrocytes. Osteonectin is a secreted heat-shock protein.

We investigated the induction of specific protein expression by heat shock in dedifferentiated and hypertrophic chick embryo chondrocytes in a culture system that allows 'in vitro' differentiation of cartilage cells [Castagnola, P., Moro, G., Descalzi-Cancedda, F. and Cancedda, R. (1986) J. Cell. Biol. 102, 2310-2317]. As control, we used cultures of embryonic fibroblasts from the whole body and from the skin. In the cell lysates of all cultures we identified four major heat-shock proteins (HSP), with a molecular size corresponding to HSP families previously described (HSP 90, HSP 70, HSP 47 and HSP 26). Some of these proteins were constantly induced when the temperature was raised, others were expressed in a more variable manner. Differences also existed in the relative amount of the HSP synthesized by the four cultures. When we specifically investigated HSP species released into the culture medium, we observed a 43-45 kDa protein constantly expressed and secreted in large amount by the cells. On the basis of its biochemical characteristic and its precipitation by specific antibodies, this protein has been identified as osteonectin (SPARC, BM-40).

Isolation of a novel bone glycosylated phosphoprotein with disulphide cross-links to osteonectin.

An 80 kDa protein was purified from calf bone by HCl-demineralization followed by 0.5 M EDTA/1.0 M NaCl extraction and sequential chromatography on DE-52, hydroxyapatite, and TSK-gel G3000SW HPLC columns. From the DE-52 column the protein was eluted at three different fractions, of which one further separated into two fractions on the hydroxyapatite column, indicating that the protein is present in four different molecular forms designated as 80 k-I-1, k-I-2, k-II, k-III. The N-terminal sequence analysis of all four forms gave the same sequence, SEQYNQEPNNV. Several tryptic internal peptides were also generated, purified and sequenced, leading to the identification of several repeat sequences, IFLGXXEI. Homology searching of the N-terminal and internal sequences indicates that this is a novel protein. Both 80 k-I-2 and k-III had similar amino acid composition with high contents of Asx, Glx and Leu and contained 7 and 16 phosphoserines per 1000 total amino acids, respectively. The 80 k-I-1 and 80 k-II forms were stained with Rhodamine B specific for phosphoproteins. The four forms contained different contents of neutral sugars ranging from 5.5 to 26% (w/w protein) and approximately 1.7% sialic acid. These data indicated that the 80 kDa protein exists in four isomeric forms, at least based on the different post-translational modifications. The evaluation of the 80 kDa glycosylated phosphoprotein under alkylating, reducing and non-reducing conditions indicated that this protein undergoes polymerization through intermolecular disulphide bonds. Furthermore, the 80 kDa protein and osteonectin (ON), both of which are cysteine-rich proteins, can cross-link with each other via disulphide bonds, and this process can be induced to take place in vitro under experimental conditions. The occurrence of such a phenomenon in vivo was confirmed from the presence of similar high Mr components containing both 80 kDa and ON in the same SDS/PAGE bands, detected by the respective antibody reactions in crude bone extracts which were extracted in the presence of alkylating agent.

Structural variability of BM-40/SPARC/osteonectin glycosylation: implications for collagen affinity.

We performed a detailed investigation of N-glycan structures on BM-40 purified from different sources including human bone, human platelets, mouse Engelbreth-Holm-Swarm (EHS) tumor, and human BM-40 recombinantly expressed in 293 and osteosarcoma cells. These preparations were digested with endoglycosidases and N-glycans were further characterized by sequential exoglycosidase digestion and high-performance liquid chromatography (HPLC) analyses. Bone BM-40 carries high-mannose structures as well as biantennary complex type N-glycans, whereas the protein from platelets and 293 cells has exclusively bi- and triantennary complex type structures. BM-40 derived from the EHS tumor carries biantennary complex type and additional hybrid structures. Using the osteosarcoma-derived MHH-ES1 cell line we successfully expressed a recombinant BM-40 that bears at least in part the bone-specific high-mannose N-glycosylation in addition to complex type and hybrid structures. Using chromatography on Concanavalin-A Sepharose, we further purified a fraction enriched in high-mannose structures. This array of differentially glycosylated BM-40 proteins was assayed by surface plasmon resonance measurements to investigate the binding to collagen I. BM-40 carrying high-mannose structures binds collagen I with higher affinity, suggesting that differentially glycosylated forms may have different functional roles in vivo.

SPARC induces the expression of type 1 plasminogen activator inhibitor in cultured bovine aortic endothelial cells.

SPARC, a Ca(2+)-binding glycoprotein that is expressed during tissue morphogenesis and functions as an inhibitor of cell spreading in vitro, was found to induce the secretion of an Mr = 45,000 protein in bovine aortic endothelial (BAE) cells. This protein was identified as type 1 plasminogen activator inhibitor (PAI-1) on Western blots with anti-PAI-1 antiserum. SPARC stimulated the secretion of PAI-1 protein into the medium of subconfluent BAE cells, but not confluent BAE cells, in a dose- and time-dependent manner. Secretion of PAI-1 into the culture medium was progressive and exhibited an increase of 3- to 7-fold over control values within 24 h after the addition of SPARC. Levels of PAI-1 mRNA were elevated 2-fold within 4 to 24 h after the addition of SPARC and did not increase with higher concentrations of SPARC. Since the induction of PAI-1 mRNA by SPARC was not blocked by cycloheximide, de novo protein synthesis was apparently not required for this stimulation. Control experiments showed that the induction of PAI-1 was not due to contamination of the SPARC preparations with endotoxin. These data demonstrate that SPARC induces the biosynthesis of PAI-1 in BAE cells and suggest a role for SPARC in the regulation of fibrinolysis and in the control of proteolytic events in remodeling tissues.

Role of N-linked glycosylation in human osteonectin. Effect of carbohydrate removal by N-glycanase and site-directed mutagenesis on structure and binding of type V collagen.

In this study we demonstrate that the binding region of recombinant truncated human bone osteonectin (tHON) for type V collagen resides between amino acids 1 and 146. After removal of oligosaccharide chain structures from tHON, bovine bone osteonectin (BBON) and human platelet osteonectin (HPON) by N-glycanase, their ability to bind to type V collagen is increased, and HPON affinity to collagen V is the same as that of BBON. These data suggest that glycosylation of osteonectin has a direct or regulatory effect on osteonectin binding to collagen V and that the increase in tHON binding upon removal of carbohydrate is the result of a loss of a down-regulation site or direct interference of the carbohydrate at the binding site. To determine the specific role of each N-glycosylation site in tHON, Asn71 and Asn99 were mutated to Gln (N71Q, N99Q) and Thr73 and Thr101 mutated to Ala (T73A, T101A) to selectively inhibit oligosaccharide attachment. The binding affinity of N99Q and T101Q to collagen V is markedly increased over wild-type tHON, whereas N71Q and T73A are the same as wild-type tHON. The doubled mutant (N71,99Q) binds identically to collagen V as N99Q and T101A. These data suggest that only the position 99 glycosylation site (Asn99-X-Thr101) in tHON is important in the reduction of binding of osteonectin to collagen V. Consistent with the binding data is the observation that both the N71Q and T73A mutant proteins migrate on SDS-polyacrylamide gel electrophoresis gels identically to wild-type tHON, suggesting that there is little or no N-glycosylation of residue 71 in wild-type osteonectin.

Stimulation of motility of human renal cell carcinoma by SPARC/Osteonectin/BM-40 associated with type IV collagen.

SPARC is known to be important in development and tissue remodelling. Here, we examined the effects of SPARC (secreted protein, acidic and rich in cysteine; osteonectin) derived from a rat osteosarcoma cell line on migration of renal cell carcinoma (RCC) by a Boyden chamber assay. YCR RCC cells migrated through type IV collagen-coated filters without stimuli (basal level). SPARC in the lower compartment stimulated chemotactic activity to 120% of the basal level, whereas premixing of YCR with purified SPARC before inoculation reduced their migration to 72% of the basal level. Furthermore, SPARC mixed with type IV collagen more efficiently stimulated their migration in a concentration-dependent manner (up to 170% of the basal level). This suggests that SPARC bound to type IV collagen plays a role in tumor invasion.