The identification and characterization of collagen receptors involved in HeLa cell-substratum adhesion

Four proteins of molecular mass 102, 87, 45, and 38 kDa were isolated from plasma membrane preparations by affinity chromatography. The 102-, 87-, and 38-kDa proteins were shown to be collagen receptors involved in the adhesion of HeLa cells to a gelatin substratum. All four proteins were eluted by high salt from affinity columns made of either types I or IV collagen or type I gelatin. Generally, a total of six major proteins were found in the high salt eluates, although the relative amounts of each varied among experiments. Immunoprecipitation, immunoblotting, and limited peptide mapping indicated that the 102-kDa protein was most sensitive to proteolysis leading to the formation of proteins of molecular mass 58 and 54 kDa. Even in the presence of a mixture of protease inhibitors the 58-kDa fragment was usually the more abundant species. Lectin binding indicated that the 102-, 87-, and 38-kDa proteins contain carbohydrate. Phase-partitioning with Triton X-114 and the need to solubilize the proteins in Triton X-100 indicated that the 102-, 87-, 45-, and 38-kDa proteins have a hydrophobic domain. The 87-kDa protein partitioned exclusively with the detergent-rich phase, suggesting that it is the most hydrophobic. Cell surface labeling with 125I indicated that the four proteins have an extracellular domain. Four criteria were used to determine which of the four proteins are collagen receptors mediating cell-substrate adhesion: 1) during HeLa cell adhesion, proteins with Mr values similar to all four proteins or their peptide fragments were cross-linked to a gelatin substratum derivatized with a photoactivatable probe; 2) a pentapeptide containing the Arg-Gly-Asp cell recognition sequence eluted the same four proteins as those found by high salt elution of collagen affinity columns; 3) monospecific antibodies to the 102-, 87-, and 38-kDa proteins, but not the 45-kDa protein, inhibited the spreading of HeLa cells on a gelatin substratum; 4) monospecific antibodies to the 102-, 87-, and 38-kDa proteins, but not the 45-kDa protein, bound to culture dishes substituted for gelatin in mediating the spreading of HeLa cells. Taken together, the data suggest that the 102-, 87-, and 38-kDa proteins are collagen receptors involved in HeLa cell adhesion. Although the 45-kDa protein has two of the characteristics of a collagen receptor defined here, it does not fit the criteria for one involved in cell-substratum adhesion.     

A collagen/gelatin-binding decapeptide derived from bovine propolypeptide of von Willebrand factor.

Propolypeptide of von Willebrand factor (pp-vWF) binds to type I collagen, and we have reported that a binding domain exists in a 21.5/21-kDa fragment originated from the C-terminal portion [Takagi, J., Fujisawa, T., Sekiya, F., & Saito, Y. (1991) J. Biol. Chem. 266, 5575-5579]. The collagen-binding property of the 21.5/21-kDa fragment was compared with that of the intact pp-vWF. Although pp-v WF preferentially binds to native type I collagen fibrils, the isolated fragment no longer has this specificity and binds well to collagen of other types in the native and heat-denatured states. In order to determine the critical site that mediates this collagen/gelatin binding, several peptides were synthesized based on the primary structure of the 21.5/21-kDa fragment. Among these, a 25-residue peptide strongly inhibited the binding of the 125I-labeled 21.5/21-kDa fragment to collagen. Using this inhibitory effect as an index, the binding site was defined to the sequence as follows: WREPSFCALS. Furthermore, a decapeptide of this sequence bound to collagen and gelatin, indicating that this sequence is responsible for the binding of the 21.5/21-kDa fragment to collagen/gelatin.     

A cell surface receptor complex for collagen type I recognizes the Arg- Gly-Asp sequence

To isolate collagen-binding cell surface proteins, detergent extracts of surface-iodinated MG-63 human osteosarcoma cells were chromatographed on affinity matrices of either type I collagen-Sepharose or Sepharose carrying a collagen-like triple-helical peptide. The peptide was designed to be triple helical and to contain the sequence Arg-Gly-Asp, which has been implicated as the cell attachment site of fibronectin, vitronectin, fibrinogen, and von Willebrand factor, and is also present in type I collagen. Three radioactive polypeptides having apparent molecular masses of 250 kD, 70 kD, and 30 kD were distinguishable in that they showed affinity toward the collagen and collagen-like peptide affinity columns, and could be specifically eluted from these columns with a solution of an Arg-Gly-Asp-containing peptide, Gly-Arg-Gly-Asp-Thr-Pro. These collagen-binding polypeptides associated with phosphatidylcholine liposomes, and the resulting liposomes bound specifically to type I collagen or the collagen-like peptide but not to fibronectin or vitronectin or heat-denatured collagen. The binding of these liposomes to type I collagen could be inhibited with the peptide Gly-Arg-Gly-Asp-Thr-Pro and with EDTA, but not with a variant peptide Gly-Arg-Gly-Glu-Ser-Pro. We conclude from these data that these three polypeptides are membrane molecules that behave as a cell surface receptor (or receptor complex) for type I collagen by interacting with it through the Arg-Gly-Asp tripeptide adhesion signal. The lack of binding to denatured collagen suggests that the conformation of the Arg-Gly-Asp sequence is important in the recognition of collagen by the receptor complex.     

Isolation and characterization of rat plasma fibronectin.

Rat plasma fibronectin has been isolated and characterized and monospecific antibodies were prepared to it. Two components of fresh rat plasma (in the presence of proteinase inhibitors) bound to a gelatin-Sepharose affinity column. One protein was eluted with 4.0 M-urea and was identified as fibronectin. Another protein was eluted from the gelatin-Sepharose column with 8.0 M-urea and was identified as a 70 000-Mr collagen-binding molecule. This 70 000-Mr fragment was found to be a normal constituent of blood plasma, and its presence did not represent a proteolytic degradation product formed during isolation. The antibodies prepared against rat fibronectin only weakly cross-reacted with plasma fibronectins of chicken, horse and human. These studies shed light on the metabolic interrelationships between fibronectin and other collagen-binding molecules.     

Spreading of HeLa cells on a collagen substratum requires a second messenger formed by the lipoxygenase metabolism of arachidonic acid released by collagen receptor clustering.

HeLa cells attach to a variety of substrata but spread only on collagen or gelatin. Spreading is dependent on collagen-receptor upregulation, clustering, and binding to the cytoskeleton. This study examines whether second messengers are involved in initiating the spreading process on gelatin. The levels of cytosolic free calcium ([Ca++]i), cAMP, and cytoplasmic pH (pHi) do not change during cell attachment and spreading. However, a basal level of [Ca++]i and an alkaline pH(i) are required for spreading. There is an activation of protein kinase C (PKC) and a release of arachidonic acid (AA) on attachment and before cell spreading. Inhibition of PKC does not block cell spreading, indicating that PKC activation is not essential for spreading. Inhibition of phospholipase A2 blocks cell spreading, whereas addition of exogeneous AA overcomes this inhibitory effect. Among AA metabolic pathways, inhibitors of lipoxygenase (LOX) block cell spreading, suggesting that a LOX product(s) formed from AA initiates spreading. Clustering receptors for collagen with polyclonal antibodies, or with anti-collagen-receptor antigen-binding fragments (Fab) in combination with a secondary antibody, induce AA release. Also, AA is released when cells attach to either immobilized gelatin or immobilized Arg-Gly-Asp (RGD) peptide. Thus, AA is released whenever receptor clustering is observed. Receptor occupancy is not sufficient to release AA; when cells are treated with gelatin or RGD peptide in solution or anti-collagen-receptor Fab fragments without secondary antibody, conditions where receptor clustering is not observed, AA is not released. Thus, a LOX metabolite(s) of AA formed by collagen-receptor clustering is a second messenger(s) that initiates HeLa cell spreading. LOX inhibitors also block the spreading of bovine aortic endothelial cells, chicken embryo fibroblasts, and CV-1 fibroblasts on gelatin or fibronectin, indicating that other cells might use the same second messenger system in initiating cell-substratum adhesion.     

Identification of integrin collagen receptors on human melanoma cells

Integrin receptors may mediate the adhesion of cells to a number of extracellular matrix components. We found that the attachment of human melanoma cells to collagen types I and IV was blocked by antibodies to the integrin beta 1 subunit but not by peptides containing the Arg-Gly-Asp sequence. Ligand affinity chromatography was used to search for integrin-related receptors which mediate adhesion to native collagens. Detergent extracts of surface 125I-iodinated melanoma cells were chromatographed on type I or IV collagen-Sepharose columns. Bound material was eluted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. EDTA, but not Arg-Gly-Asp peptides, eluted a mixture of two integrin-related heterodimeric complexes. Each complex contained the integrin beta 1 chain with Mr of 110,000 and a distinct alpha chain with Mr of either 200,000 or 150,000. Immunoprecipitation with specific monoclonal antibodies identified the complexes as very late activation antigen (VLA)-1 (alpha 1 beta 1) and VLA-2 (alpha 2 beta 1), respectively. The binding of these receptors to collagen appeared to be specific because they failed to be retained on fibronectin- or laminin-Sepharose columns. Immunofluorescent staining of cells on collagen substrates with antibodies to VLA-1 and VLA-2 localized these complexes in vinculin-positive adhesion plaques. In contrast, the receptor complexes were not detected in adhesion plaques of cells attached to fibronectin- or laminin-coated substrates. These results indicate that melanoma cells express at least two different integrin-related collagen-binding receptor complexes that appear to mediate cell adhesion to collagen.     

Studies of extracellular fibronectin matrix formation with fluoresceinated fibronectin and fibronectin fragments

Fluorescein isothiocyanate conjugated human plasma fibronectin, 70-kDa collagen-binding, 60-kDa central, 60-kDa heparin-binding, 180-kDa heparin, collagen-binding fibronectin fragments and gelatin were used to study extracellular fibronectin matrix formation. Exogenous fibronectin, gelatin, 70-kDa collagen-binding and 180-kDa heparin, collagen-binding fragments were shown to be able to bind specifically to preexisting extracellular matrix of living fibroblasts. The results suggest that: (i) Fibronectin matrix formation may occur through a self-assembly process; (ii) the NH2-terminal part of fibronectin is responsible for fibronectin-fibronectin interaction during fibronectin fibril formation; (iii) plasma fibronectin may be the source for tissue fibronectin.     

Arachidonate initiated protein kinase C activation regulates HeLa cell spreading on a gelatin substrate by inducing F-actin formation and exocytotic upregulation of β1 Integrin

HeLa cell spreading on a gelatin substrate requires the activation of protein kinase C (PKC), which occurs as a result of cell-attachment-induced activation of phospholipase A2 (PLA2) to produce arachidonic acid (AA) and metabolism of AA by lipoxyginase (LOX). The present study examines how PKC activation affects the actin- and microtubule-based cytoskeletal machinery to facilitate HeLa cell spreading on gelatin. Cell spreading on gelatin is contingent on PKC induction of both actin polymerization and microtubule-facilitated exocytosis, which is based on the following observations. There is an increase in the relative content of filamentous (F)-actin during HeLa cell spreading, and treating HeLa cells with PKC-activating phorbol esters such as 12-O-tetradecanoyl phorbol 13-acetate (TPA) further increases the relative content of F-actin and the rate and extent to which the cells spread. Conversely, inhibition of PKC by calphostin C blocked both cell spreading and the increase of F-actin content. The increased F-actin content induced by PKC activators also was observed in suspension cells treated with TPA, and the kinetics of F-actin were similar to that for PKC activation. In addition, PKCϵ, which is the PKC isoform most involved in regulating HeLa cell spreading in response to AA production, is more rapidly translocated to the membrane in response to TPA treatment than is the increase in F-actin. Blocking the activities of either PLA2 or LOX inhibited F-actin formation and cell spreading, both of which were reversed by TPA treatment. This result is consistent with AA and a LOX metabolite of AA as being upstream second messengers of activation of PKC and its regulation of F-actin formation and cell spreading. PKC appears to activate actin polymerization in the entire body of the cell and not just in the region of cell-substrate adhesion because activated PKC was associated not only with the basolateral plasma membrane domain contacting the culture dish but also with the apical plama membrane domain exposed to the culture medium and with an intracellular membrane fraction. In addition to the facilitation of F-actin formation, activation of PKC induces the exocytotic upregulation of β1 integrins from an intracellular domain to the cell surface, possibly in a microtubule-dependent manner because the upregulation is inhibited by Nocodazole. The results support the concept that cell-attachment-induced AA production and its metabolism by LOX results in the activation of PKC, which has a dual role in regulating the cytoskeletal machinery during HeLa cell spreading. One is through the formation of F-actin that induces the structural reorganization of the cells from round to spread, and the other is the exocytotic upregulation of collagen receptors to the cell surface to enhance cell spreading. J. Cell. Physiol. 173:361–370, 1997. © 1997 Wiley-Liss, Inc.     

Cdc42 and RhoA are differentially regulated during arachidonate-mediated HeLa cell adhesion

Cell adhesion to extracellular matrix requires stimulation of an eicosanoid signaling pathway through the metabolism of arachidonate by 5-lipoxygenase to leukotrienes and cyclooxygenase-1/2 to prostaglandins, as well as activation of the small GTPase signaling pathway involving Cdc42 and Rho. These signaling pathways direct remodeling of the actin cytoskeleton during the adhesion process, specifically the polymerization of actin during cell spreading and the bundling of actin filaments when cells migrate. However, few studies linking these signaling pathways have been described in the literature. We have previously shown that HeLa cell adhesion to collagen requires oxidation of arachidonic acid (AA) by lipoxygenase for actin polymerization and cell spreading, and cyclooxygenase for bundling actin filaments during cell migration. We demonstrate that small GTPase activity is required for HeLa cell spreading upon gelatin, and that Cdc42 is activated while Rho is downregulated during the spreading process. Using constitutively active and dominant negative expression studies, we show that Cdc42 is required for HeLa cell spreading and migration, while activated RhoA is antagonistic towards spreading. Constitutively active RhoA promotes cell migration and increases the degree of actin bundling in HeLa cells. Further, we demonstrate that activation of either the AA oxidation pathway or the small GTPase pathway cannot rescue inhibition of spreading when the alternate pathway is blocked. Our results suggest (1) both the eicosanoid signaling pathway and small GTPase activation are required during HeLa cell adhesion, and (2) these signaling pathways converge to properly direct remodeling of the actin cytoskeleton during HeLa cell spreading and migration upon collagen.     

Vitronectin and type-I collagen binding by Staphylococcus aureus and coagulase-negative staphylococci

Abstract Binding of ¹²⁵I-labelled type-I collagen and ¹²⁵I-labelled vitronectin (human serum spreading factor or S-protein) was studied using Staphylococcus aureus and coagulase-negative staphylococci of different species. Binding of collagen and vitronectin was time dependent for S. aureus ISP 546, and S. haemolyticus E 2498/86. Co-operative binding of vitronectin and collagen by staphylococcal cells was demonstrated. Binding to S. haemolyticus E 2498/86 was more rapid and was enhanced in vitronectin/collagen mixtures than for either protein separately. Furthermore, pre-incubation of staphylococcal cells with unlabelled collagen enhanced vitronectin binding. When cells of S. haemolyticus E 2498/86 were treated with pronase E, proteinase K, subtilopeptidase A or trypsin, vitronectin-binding was decreased by 50% or more, whereas collagen-binding was protease resistant. For the strains of S. aureus tested, both vitronectin and collagen binding were found to be protease sensitive. Type-I collagen peptides inhibited collagen-binding to S. haemolyticus E 2498/86, whereas vitronectin-binding was not affected perhaps indicating different receptors for these proteins. The binding of both collagen and vitronectin was shown to be reversible, since bound ¹²⁵I-collagen and ¹²⁵I-vitronectin were displaced after adding excess of the homologous protein.     

Triflavin, an Arg-Gly-Asp-containing peptide, inhibits human cervical carcinoma (HeLa) cell-substratum adhesion through an RGD-dependent mechanism

Triflavin, a 7.5-kDa cysteine-rich polypeptide purified from Trimeresurus flavoviridis snake venom, belongs to a family of RGD-containing peptides, termed disintegrins, that have been isolated from the venoms of various vipers and shown to be potent inhibitors of platelet aggregation. The interaction of tumor cells with extracellular matrices such as fibronectin, vitronectin, and collagen has been shown to be mediated through a family of cell surface receptors that specifically recognize an arginine-glycine-aspartic acid (RGD) sequence within each adhesive protein. In this study, we show that triflavin dose-dependently inhibited adhesion of human cervical carcinoma (HeLa) cells to extracellular matrices (ECMs; i.e., fibronectin, fibrinogen, and vitronectin). On the other hand, triflavin exerted a limited inhibitory effect on cell adhesion to laminin and collagen (type I and IV). On a molar basis, triflavin is approximately 800 times more potent than Gly-Arg-Gly-Asp-Ser (GRGDS) at inhibiting cell adhesion. When immobilized on plate, triflavin significantly promoted HeLa cell adhesion, and this attachment was inhibited by GRGDS. Furthermore, FITC-conjugated triflavin bound to cells in a saturable manner and its binding was inhibited by GRGDS. In addition, triflavin did not affect [³H]thymidine uptake of HeLa cells during a 3-day incubation. These results suggest that triflavin probably binds to integrin receptors expressed on HeLa cell surface via its RGD sequence within its molecule, thereby inhibiting the adhesion of extracellular matrices to HeLa cells.     

Production of a biologically active epidermal growth factor fusion protein with high collagen affinity

Collagen is generally incapable of capturing polypeptides such as growth factors in a specific manner. In this study, we established a collagen-binding growth factor (FNCBD-EGF) consisting of epidermal growth factor (EGF) and the fibronectin collagen-binding domain. A typical yield of FNCBD-EGF was approximately 200 microg/ml culture in an Escherichia coli expression system. This fusion protein bound to gelatin and fibrillar collagen sponges, and the bound protein was not effectively eluted even with 2 M NaCl. In addition, FNCBD-EGF bound to type I, II, III, or IV collagen-coated plates, and the specificity of binding was confirmed by competitive inhibition using fibronectin. FNCBD-EGF substantially stimulated cell growth after binding to collagen-coated culture plates, whereas EGF had no effect, indicating that this fusion protein acted as a collagen-associated growth factor. In an animal model of impaired wound healing, FNCBD-EGF, but not EGF, was retained with collagen sponges at wound sites 4 d after implantation, and repair of epidermis was observed underneath the sponges. These results suggested that our fusion protein with high collagen affinity would be useful for wound healing.     

A collagen-binding protein involved in the differentiation of myoblasts recognizes the Arg-Gly-Asp sequence

We had earlier demonstrated that a 46-kDa glycoprotein is involved in the differentiation of rat skeletal myoblasts. We now show that the binding of this glycoprotein to collagen and gelatin is disrupted by Arg-Gly-Asp (RGD) containing peptide but not by Arg-Gly-Glu (RGE). The former peptide also selectively elutes the 46-kDa glycoprotein bound to gelatin-Sepharose. Since all other proteins which bind RGD sequences have been found at the cell surface, we attempted to localize the 46-kDa glycoprotein by means of immuno fluorescent staining and radioiodine labeling. Surprisingly, the majority of the protein was found to be localized in the endoplasmic reticulum. Protease treatment of a microsomal fraction revealed that the protein is in the interior of the reticulum. Immunoprecipitation experiments, using a polyclonal antibody against the 46-kDa protein, demonstrated that no closely related proteins exist in myoblasts and also confirmed that the protein was not a fragment of a cell-surface localized protein. These findings suggest that the RGD sequence is also used in protein recognition within the cell.     

Identification and distribution of a novel, collagen-binding protein in the developing subepicardium and endomysium.

A protein doublet (M(r) = 68,000) that copurifies with chicken cardiac collagen types I and III is purified and characterized in the present study. Peptide mapping and amino terminus sequencing for both 68-kDa polypeptides show they have similar structures. This is supported by amino terminus sequencing of a 39-kDa proteolytic fragment of each polypeptide. The 68-kDa polypeptides appear at pI 6.7-6.8 in two-dimensional gels. Under nonreducing, electrophoretic conditions, the doublet appears as a large multimer or aggregate. Amino acid sequencing of the protein shows that its amino terminus contains a heptapeptide (VCLXXGK) that appears in the heparin/fibrin-binding domain of fibronectin and the collagen-binding domain of laminin. Cardiac myocytes synthesize and secrete the protein in vitro onto cell surfaces and onto the substratum. Indirect immunofluorescence shows the protein first appears in the chicken subepicardium at approximately 10 days following fertilization. As collagen accumulates in the subepicardium and the volume of the subepicardial space increases, the 68-kDa protein is found predominantly at the interface between myocardial cells and the connective tissue and between epicardial cells and the connective tissue. In adult hearts, the protein is also present at lower concentrations in endomysial connective tissue. The 68-kDa protein is also present in the skeletal muscle endomysium of embryonic chickens. Electron microscopic immunocytochemistry shows the 68-kDa protein is located at the surface of subepicardial collagen fibers. In addition, a direct interaction between the 68-kDa protein and collagen are indicated by: 1) equilibrium gel filtration of the 68-kDa protein in the presence of gelatin, 2) gelatin affinity chromatography of the 68-kDa protein, and 3) comigration of type I collagen and the 68-kDa protein during gel filtration under reducing conditions. The 68-kDa protein exhibits no collagenase activity under native conditions or in zymograms. Together, the data indicate that the 68-kDa protein is a novel collagen-associated protein appearing in late epicardial development.     

Fibronectin is not a serum spreading factor for HeLa cells

The spreading of HeLa cells on plastic substratum is mediated by fibronectin-depleted foetal calf serum but not by fibronectin isolated by gelatin-Sepharose affinity chromatography. The same is true for freshly explanted chick embryonic chondrocytes. In contrast, BHK cell spreading exceeds 67% after 120 min at 37 degrees C in fibronectin-supplemented (10 micrograms ml-1) serum-free medium. Long-term cultivation of HeLa cells in Eagle's MEM supplemented with fibronectin-free serum is associated with the accumulation of cells in mitosis or before cytokinesis; many cells die and the remaining living cells, characterized by marked changes in morphology, multiply very slowly. It can be concluded therefore that fibronectin does not produce spreading in HeLa cells but forces them into mitosis.     

Identification of collagen-binding proteins in Lactobacillus spp. with surface-enhanced laser desorption/ionization-time of flight ProteinChip technology

Biosurfactants produced by Lactobacillus fermentum RC-14, L. rhamnosus GR-1 and 36, and L. casei Shirota were found to contain proteins that bind to both collagen types III and VI, as determined by surface-enhanced laser desorption/ionization (SELDI)-time of flight mass spectrometry. Both collagen types III and VI immobilized on SELDI preactivated ProteinChip arrays detected several different sizes (2 to 48 kDa) of collagen-binding proteins. Overall, the RC-14-produced biosurfactant contained the greatest number of collagen-binding proteins (RC-14 > GR-1 > 36 > Shirota), including the mature form of a previously cloned 29-kDa collagen-binding protein (referred to in its mature 26-kDa form). Although biosurfactants isolated from L. casei Shirota and L. rhamnosus 36 and GR-1 also contain several collagen-binding proteins, they do not contain the 26-kDa collagen-binding protein. Together, these results demonstrate the utility of the SELDI system as a means of rapidly characterizing clinically important but complex biosurfactant solutions.     

Developmental regulation of a secreted gelatin-binding protein during myogenesis in vitro

Collagen has a stimulatory effect on the differentiation of skeletal muscle cells in culture. Putative collagen-binding proteins were isolated from detergent-solubilized cultures of the L6 rat muscle cell line and primary clonal cultures of human skeletal muscle satellite cells, using gelatin-Sepharose affinity chromatography. In addition to fibronectin, which has been reported by others to be synthesized by cultured muscle cells, we found that muscle cultures synthesized gelatin-binding proteins of lower apparent molecular weight. Only one of these proteins was secreted into the growth medium and bound to type I collagen. Binding of this protein to gelatin and collagen-Sepharose was resistant to repeated washing with 1 M NaCl and nonionic detergent. The secreted gelatin-binding protein had an apparent molecular weight of 63,000-72,000, depending upon the conditions of electrophoresis. The lack of reactivity of the secreted protein with polyclonal antisera against fibronectin, the lack of effect of protease inhibitors on its appearance in the medium, and the rapid de novo production of the protein during pulse labeling with radioactive methionine indicated that it was not a fibronectin fragment. The rate of synthesis of the secreted gelatin-binding protein increased markedly during the myogenesis of rat and human cultures.     

The cell adhesion domain of type XVII collagen promotes integrin-mediated cell spreading by a novel mechanism

Type XVII collagen (BP180) is a keratinocyte transmembrane protein that exists as the full-length protein in hemidesmosomes and as a 120-kDa shed ectodomain in the extracellular matrix. The largest collagenous domain of type XVII collagen, COL15, has been described previously as a cell adhesion domain (Tasanen, K., Eble, J. A., Aumailley, M., Schumann, H., Baetge, J, Tu, H., Bruckner, P., and Bruckner-Tuderman, L. (2000) J. Biol. Chem. 275, 3093-3099). In the present work, the integrin binding of triple helical, human recombinant COL15 was tested. Solid phase binding assays using recombinant integrin alpha(1)I, alpha(2)I, and alpha(10)I domains and cell spreading assays with alpha(1)beta(1)- and alpha(2)beta(1)-expressing Chinese hamster ovary cells showed that, unlike other collagens, COL15 was not recognized by the collagen receptors. Denaturation of the COL15 domain increased the spreading of human HaCaT keratinocytes, which could migrate on the denatured COL15 domain as effectively as on fibronectin. Spreading of HaCaT cells on the COL15 domain was mediated by alpha(5)beta(1) and alpha(V)beta(1) integrins, and it could be blocked by RGD peptides. The collagen alpha-chains in the COL15 domain do not contain RGD motifs but, instead, contain 12 closely related KGD motifs, four in each of the three alpha-chains. Twenty-two overlapping, synthetic peptides corresponding to the entire COL15 domain were tested; three peptides, all containing the KGD motif, inhibited the spreading of HaCaT cells on denatured COL15 domain. Furthermore, this effect was lost by mutation from D to E (KGE instead of KGD). We suggest that the COL15 domain of type XVII collagen represents a specific collagenous structure, unable to interact with the cellular receptors for other collagens. After being shed from the cell surface, it may support keratinocyte spreading and migration.     

Recombinant collagen for animal product-free dextran microcarriers

Manufacturers of vaccines and other biologicals are under increasing pressure from regulatory agencies to develop production methods that are completely animal-component-free. In order to comply with this demand, alternative cell culture substrates to those now on the market, primarily collagen or gelatin, must be found. In this paper, we have tested a number of possible substitutes including recombinant collagen, a 100-kDa recombinant gelatin fragment and a peptide derived from a cell-binding region of type I collagen. The small 15-amino acid peptide did not support attachment of human fibroblasts in monolayer culture. The 100-kDa gelatin fragment supported cell attachment in monolayer culture, but was significantly less active than intact porcine gelatin. Recombinant type I collagen was as successful in promoting cell attachment as native collagen, and both were more effective than porcine gelatin. Based on these data, dextran microspheres were treated with the same attachment proteins—porcine gelatin, native collagen, or recombinant collagen. The same trends were observed as in monolayer culture. Concentrations of the recombinant collagen (as well as native collagen) supported cell attachment on dextran microspheres at concentrations as low as 0.01 μg/cm². Treatment of the dextran with a low level of polyethylenimine, a cationic moiety, further enhanced attachment when used in conjunction with the low concentration of recombinant collagen. Where there was increased cell attachment, increased proliferation followed. We are confident, based on these findings, that a fully recombinant substitute could replace gelatin in current microcarrier preparations without losing the cell growth benefits provided by the native protein.     

Mapping of SPARC/BM-40/osteonectin-binding sites on fibrillar collagens

The 33-kDa matrix protein SPARC (BM-40, osteonectin) binds several collagen types with moderate affinity. The collagen-binding site resides in helix alphaA of the extracellular calcium-binding domain of SPARC and is partially masked by helix alphaC. Previously, we found that the removal of helix alphaC caused a 10-fold increase in the affinity of SPARC for collagen, and we identified amino acids crucial for binding by site-directed mutagenesis. In this study, we used rotary shadowing, CNBr peptides, and synthetic peptides to map binding sites of SPARC onto collagens I, II, and III. Rotary shadowing and electron microscopy of SPARC-collagen complexes identified a major binding site approximately 180 nm from the C terminus of collagen. SPARC binding was also detected with lower frequency near the matrix metalloproteinase cleavage site. These data fit well with our analysis of SPARC binding to CNBr peptides, denaturation of which abolished binding, indicating triple-helical conformation of collagen to be essential. SPARC binding was substantially decreased in two of seven alpha2(I) mutant procollagen I samples and after N-acetylation of Lys/Hyl side chains in wild-type collagen. Synthetic peptides of collagen III were used to locate the binding sites, and we found SPARC binding activity in a synthetic triple-helical peptide containing the sequence GPOGPSGPRGQOGVMGFOGPKGNDGAO (where O indicates 4-hydroxyproline), with affinity for SPARC comparable with that of procollagen III. This sequence is conserved among alpha chains of collagens I, II, III, and V. In vitro collagen fibrillogenesis was delayed in the presence of SPARC, suggesting that SPARC might modulate collagen fibril assembly in vivo.