Functional differences in the interactions of glycosylation-deficient cell lines with fibronectin, laminin, and type IV collagen

Fibronectin isolated from the conditioned medium of monolayer cultures of baby hamster kidney (BHK) cells and several ricin-resistant (Ric) mutants derived from them express differences in N-glycosylation. The asparagine-linked oligosaccharides of BHK cell-derived fibronectin consist largely of complex chains, whereas hybrid and/or high-mannose chains are present in the fibronectins of mutant cell lines. The fibronectins exhibiting different glycosylation patterns are incorporated to similar extents into the cell-layer of human skin fibroblasts. In contrast, mutant cells retain significantly less endogenously produced fibronectin than BHK cells and also incorporate less human cellular fibronectin into a pericellular matrix. In vitro adhesion assays show that mutant cells attach to and spread relatively poorly on fibronectin-or type IV collagen-coated substrata but interact as well as do BHK cells with a laminin substratum. These results indicate that asparagine-linked oligosaccharides of fibronectin are not required for the binding and incorporation of the molecule into cell layers, but, as constituents of other cellular glycoproteins, they do modulate the ability of BHK cells to interact with some matrix components.     

Functional integrins from normal and glycosylation-deficient baby hamster kidney cells. Terminal processing of asparagine-linked oligosaccharides is not correlated with fibronectin-binding activity.

We have examined the properties of the alpha 5 beta 1 integrin of baby hamster kidney (BHK) cells, a ricin-resistant variant Ric14 lacking N-acetylglucosaminyl transferase I, and hence unable to complete assembly of hybrid- or complex-type N-glycans, and BHK cells treated with 1-deoxymannojirimycin (dMM), an inhibitor of Golgi mannosidases involved in the initial processing of N-glycan precursors. Comparable amounts of alpha 5 beta 1 integrin were isolated from these cells by chromatography of detergent extracts on a fibronectin cell-binding fragment affinity column and elution with EDTA. The alpha 5 beta 1 integrin obtained from normal BHK cells by fibronectin affinity chromatography contained mainly endoglycosidase H-resistant oligosaccharides, whereas in RicR14 cells or dMM-treated BHK cells these were entirely endoglycosidase H-sensitive. Analysis of lactoperoxidase labeled or long term biosynthetically 35S-labeled proteins from cultures of normal or glycosylation deficient cells showed similar steady state levels of alpha 5 beta 1 integrin and expression at the cell surface. Pulse-chase experiments in normal BHK cells showed rapid conversion of the alpha 5 subunit into a mature form containing oligosaccharides resistant to endoglycosidase H and slower maturation of a precursor beta 1 subunit, as in other cell types. In Ric14 cells the precursor beta 1 subunit was found to carry glycans larger than the fully processed Man5GlcNAc2 glycan of the mature subunit, indicating that the bulk precursor pool had not been translocated into the cis-Golgi compartment containing mannosidase I. We conclude that in BHK cells terminal oligosaccharide processing of alpha 5 beta 1 integrin subunits is not required for dimer formation, surface expression, and fibronectin binding, and that expression of the glycosylation defect of Ric14 cells on the alpha 5 beta 1 integrin does not account for the reduced adhesiveness of these cells on fibronectin compared with normal and dMM-treated BHK cells.     

Collagen can modulate cell interactions with fibronectin

We have examined the effects of soluble collagen on the function of fibronectin in baby hamster kidney (BHK) cells. Collagen and its purified alpha1(l) chain noncompetitively inhibited cell spreading on substrates precoated with fibronectin or a 75,000-D cell-binding fragment of fibronectin. Neither preincubation of cells with collagen followed by washing nor the addition of collagen to previously spread cells had any inhibitory effect on cell spreading, which indicates a requirement for the concurrent presence of collagen during the process of spreading. Treatment of collagen or alpha1(l) chain with collagenase abolished the inhibitory effect on fibronectin-mediated cell spreading. However, direct attachment of BHK cells to fibronectin-coated or 75,000-D fragment-coated substrates was not inhibited by collagen or by the alpha1(l) chain. Moreover, the binding of [3H]fibronectin or the 3'-75,000-D fragment to cell surfaces was not inhibited by the presence of soluble collagen, whereas soluble fibronectin inhibited binding. Although the binding of [3H]fibronectin-coated beads to BHK cell surfaces was also not inhibited by collagen, the phagocytosis of such beads was inhibited by the presence of collagen. On the other hand, soluble fibronectin partially inhibited the binding of fibronectin-coated beads but did not inhibit phagocytosis of the beads that did bind. The mechanism of the inhibition of fibronectin function by collagen and the possible interactions of two different kinds of receptors on the cell surface are discussed.     

Inhibition of fibronectin receptor function by antibodies against baby hamster kidney cell wheat germ agglutinin receptors

Previous studies suggest that the baby hamster kidney (BHK) cell fibronectin receptor is also a wheat germ agglutinin receptor (WGA-R). To analyze this possibility further, IgG and Fab fragments of antibodies produced against a BHK cell WGA-R preparation were tested to determine their effects on cell adhesion mediated by fibronectin, wheat germ agglutinin, concanavalin A, and polycationic ferritin. The WGA-R preparation was isolated by octylglucoside extraction of BHK cells followed by chromatography of the extract on WGA-agarose. The antibodies against the WGA-R preparation reacted primarily with polypeptides of molecular weights 48, 61, 83, 105, 120, 165, 210, and 230 kilodaltons (kdaltons). It was concluded that the antibodies interfered with BHK cell fibronectin receptors on the basis of the ability of anti-WGA-R IgG or Fab fragments to (a) inhibit cell spreading on fibronectin-coated substrata; (b) cause rounding and detachment of cells previously spread on fibronectin-coated substrata; and (c) inhibit binding of fibronectin-coated latex beads to the cells. Antibody activity was blocked by treatment of anti-WGA-R with the WGA-R preparation or by absorption of anti-WGA-R with intact BHK cells. The antibodies also appeared to prevent coupling of ligand-receptor complexes (involving concanavalin A or polycationic ferritin) with the cytoskeleton. Finally, cell rounding and detachment caused by the antibodies were found to require metabolic energy since it did not occur in the presence of azide or at 4 degrees C.     

Binding of plasma fibronectin to the surfaces of BHK cells in suspension at 4 °C

Plasma fibronectin is shown by several different criteria to bind to suspended BHK cells if the binding incubations are carried out at 4 °C. In indirect immunofluorescence experiments, fibronectin bound to suspended BHK cells at 4 °C in a punctate distribution over the entire cell surfaces. Little binding, however, was detected on cells incubated with fibronectin at 37 °C. The fibronectin bound to the cells at 4 °C was functionally active, since these cells subsequently were able to spread on tissue culture dishes in protein-free medium, unlike cells preincubated with fibronectin at 37 °C or in the absence of fibronectin. Also, the cell surface receptors for soluble fibronectin and fibronectin-coated beads appeared to be similar, since cells preincubated with fibronectin at 4 °C subsequently bound fewer fibronectin-coated beads than control cells. In biochemical studies with radiolabeled fibronectin, binding of fibronectin to the cells was shown to increase with incubation time up to 4 h. In competition experiments with unlabeled fibronectin, 30% of the binding of radiolabeled fibronectin could be inhibited.     

Binding specificity of a baby hamster kidney lectin for H type I and II chains, polylactosamine glycans, and appropriately glycosylated forms of laminin and fibronectin.

The carbohydrate binding specificity of Mr = 30,000 lectin (CBP30) from baby hamster kidney (BHK) cells has been studied by inhibition of binding of the radiolabeled lectin to asialofetuin-Sepharose using model oligosaccharides and glycopeptides. CBP30 binds type I or II Gal beta(1----3(4))GlcNAc chains but not Gal(beta 1----3)GalNAc. The inhibitory potency of straight chain polylactosamine structures or complex-type branched glycans is increased in proportion to the number of Gal(beta 1----3(4)) units present. Fucosylation or sialylation of terminal galactose residues or further substitution by (alpha 1----3)-linked galactose or N-acetylgalactosamine does not affect binding whereas substitution of the penultimate N-acetylglucosamine residue drastically reduces binding. Thus, blood group A, H type I or H type II structures, shows high affinity whereas Lex, Lea, and Leb structures bind poorly. CBP30 binds to murine Engelbreth-Holm-Swarm (EHS) tumor laminin and human amniotic fluid fibronectin but not human plasma fibronectin. Binding involves polylactosamine glycans as well as tri- and tetraantennary complex-type glycans present in EHS laminin and amniotic fluid fibronectin but absent in plasma fibronectin. Proteolytic fragments of EHS laminin (E1X/Nd, P1, E8, and E3) bind CBP30, but only fragment E8 supports attachment and spreading of BHK cells. BHK cell adhesion to EHS laminin or fragment E8 was not disturbed by CBP30-specific antibodies, but at relatively high concentrations (45 micrograms/ml) CBP30 inhibited spreading and partially attachment of cells on laminin.     

Analysis by lectin affinity chromatography of N-linked glycans of BHK cells and ricin-resistant mutants.

Normal baby hamster kidney (BHK) fibroblasts and ricin-resistant (RicR) mutants of BHK cells derived from them were labelled metabolically with [3H]mannose or [3H]fucose. Glycopeptides obtained by digestion of disrupted cells with Pronase were separated by affinity chromatography on concanavalin A-Sepharose. In the normal BHK cells major glycopeptide fractions were obtained consisting of tetra- and tri-antennary sialylated complex glycans, bi-antennary sialylated glycans, and neutral oligomannosidic chains. The majority of bi-antennary chains were shown to contain a fucosyl-(alpha 1-6)-N-acetylglucosaminyl sequence in the core region by their ability to bind to a lentil lectin affinity column. All of the mutant cell lines examined were found to accumulate oligomannosidic glycans in cellular glycoproteins: complex sialylated glycans were either absent or greatly reduced in amount. Analysis of fractions isolated from concanavalin A-Sepharose by Bio-Gel P-4 chromatography and glycosidase degradation indicated that the glycans accumulating in RicR14 cells have the general structure: (formula; see text) and derivatives having fewer alpha-mannosyl units. We have also analysed the glycopeptides released by trypsin treatment from the surface of the normal and mutant cells, as well as those obtained by proteolysis of fibronectin isolated from the medium. The glycopeptide profiles of the cell-surface-derived material and of fibronectin showed for the mutant cells a marked accumulation of oligomannosidic chains at the expense of complex oligosaccharide chains. Hence, the alterations in glycan structure detected in bulk cellular glycoproteins of RicR cells are expressed also in cell surface glycoproteins and in fibronectin, a secreted glycoprotein.     

Role of integrin receptors in manganese-dependent BHK cell spreading on albumin-coated substrata.

In manganese-containing medium, tissue cells can spread on albumin and other substrata typically nonadhesive for cells in calcium/magnesium-containing medium. To learn whether integrin receptors play a role in Mn-dependent adhesion, we tested the effects of RGD peptides and polyclonal anti-fibronectin receptor antibodies on BHK cell spreading on fibronectin and albumin-coated substrata. In Ca/Mg-containing medium on fibronectin substrata, the RGD-related peptides GRG-DSP and GRGDS but not RGDS inhibited cell spreading. In Mn-containing medium, spreading on albumin was inhibited by GRGDSP and GRGDS and also by RGDS. GRGESP, on the other hand, did not inhibit cell spreading under any condition tested. Antibodies directed against fibronectin receptors also inhibited Mn-dependent cell spreading on albumin substrata, but higher levels of antibody were required than were necessary to inhibit Ca/Mg-dependent spreading on fibronectin. On the basis of these results, we suggest that integrin receptors, but probably not fibronectin receptors, mediate Mn-dependent BHK cell spreading on albumin.     

Molecular requirements for the adhesion and spreading of hamster fibroblasts

Hamster plasma fibronectin adsorbs rapidly to a plastic surface to form a monolayer that promotes attachment and spreading of baby hamster kidney (BHK) fibroblasts. Maximal spreading requires a minimum surface density of about 1.8 × 10¹⁰ fibronectin molecules/cm² indicating a maximum of 45 000 contacts between a well-spread cell and the adsorbed fibronectin lattice. Surfaces coated with other antibodies or lectins reactive with the BHK cell surface also promote rapid cell flattening.     

Molecular requirements for adhesion and spreading of hamster fibroblasts.

Hamster plasma fibronectin adsorbs rapidly to a plastic surface to form a monolayer that promotes attachment and spreading of baby hamster kidney (BHK) fibroblasts. Maximal spreading requires a minimum surface density of about 1.8 × 10¹⁰ fibronectin molecules/cm² indicating a maximum of 45 000 contacts between a well-spread cell and the adsorbed fibronectin lattice. Surfaces coated with other antibodies or lectins reactive with the BHK cell surface also promote rapid cell flattening.     

Adhesion defective BHK cell mutant has cell surface heparan sulfate proteoglycan of altered properties

In the light of accumulating data that implicate cell surface heparan sulfate proteoglycans (HSPGs) with a role in cell interactions with extracellular matrix molecules such as fibronectin, we have compared the properties of these molecules in wild-type BHK cells and an adhesion-defective ricin-resistant mutant (RicR14). Our results showed that the mutant, unlike BHK cells, cannot form focal adhesions when adherent to planar substrates in the presence of serum. Furthermore, while both cell lines possess similar amounts of cell surface HSPG with hydrophobic properties, that of RicR14 cells had decreased sulfation, reduced affinity for fibronectin and decreased half-life on the cell surface when compared to the normal counterpart. Our conclusions based on this data are that these altered properties may, in part, account for the adhesion defect in the ricin-resistant mutant. Whether this results from the known alteration in assembly of N-linked glycans affecting the carbohydrate chains on the proteoglycan or some other combination of factors is discussed.     

Monoclonal antibody characterization of two distant sites required for function of the central cell-binding domain of fibronectin in cell adhesion, cell migration, and matrix assembly

Site-directed mutagenesis studies have suggested that additional peptide information in the central cell-binding domain of fibronectin besides the minimal Arg-Gly-Asp (RGD) sequence is required for its full adhesive activity. The nature of this second, synergistic site was analyzed further by protein chemical and immunological approaches using biological assays for adhesion, migration, and matrix assembly. Fragments derived from the cell-binding domain were coupled covalently to plates, and their specific molar activities in mediating BHK cell spreading were compared with that of intact fibronectin. A 37-kD fragment purified from chymotryptic digests of human plasma fibronectin had essentially the same specific molar activity as intact fibronectin. In contrast, other fragments such as an 11.5-kD fragment lacking NH2-terminal sequences of the 37-kD fragment had only poor spreading activity on a molar basis. Furthermore, in competitive inhibition assays of fibronectin-mediated cell spreading, the 37-kD fragment was approximately 325-fold more active than the GRGDS synthetic peptide on a molar basis. mAbs were produced using the 37-kD protein as an immunogen and their epitopes were characterized. Two separate mAbs, one binding close to the RGD site and the other to a site approximately 15 kD distant from the RGD site, individually inhibited BHK cell spreading on fibronectin by greater than 90%. In contrast, an antibody that bound between these two sites had minimal inhibitory activity. The antibodies found to be inhibitory in cell spreading assays for BHK cells also inhibited both fibronectin-mediated cell spreading and migration of human HT-1080 cells, functions which were also dependent on function of the alpha 5 beta 1 integrin (fibronectin receptor). Assembly of endogenously synthesized fibronectin into an extracellular matrix was not significantly inhibited by most of the anti-37-kD mAbs, but was strongly inhibited only by the antibodies binding close to the RGD site or the putative synergy site. These results indicate that a second site distant from the RGD site on fibronectin is crucial for its full biological activity in diverse functions dependent on the alpha 5 beta 1 fibronectin receptor. This site is mapped by mAbs closer to the RGD site than previously expected.     

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.     

Involvement of plasma membrane dipeptidyl peptidase IV in fibronectin-mediated adhesion of cells on collagen

Dipeptidyl peptidase IV is an exopeptidase found in the serum and in plasma membranes of most animal tissues. The role of this enzyme in cell-matrix interaction of BHK cells and hepatocytes grown on collagen-coated surfaces was investigated by three different approaches. 1) Glass surfaces were derivatized with bovine serum albumin which resulted in a cell-repulsing substratum. When it was further modified with Gly-Pro-Ala tripeptide, which is a substrate for dipeptidyl peptidase IV, BHK fibroblasts spread on it rapidly. The spreading could be inhibited by addition of free Gly-Pro-Ala or other substrates of the enzyme as well as by an inhibitor peptide Val-Pro-Leu. It was not influenced by tripeptides which were neither substrates nor inhibitors of dipeptidyl peptidase IV. 2) The addition of Gly-Pro-Ala to seeded cells slowed down the initial process of cell spreading on denatured collagen in the presence of fibronectin. The presence of both collagen and fibronectin was a necessary precondition for the spreading of cells in a manner sensitive to Gly-Pro-Ala. 3) Antiserum raised against mouse liver dipeptidyl peptidase IV added to the medium delayed the spreading of rat hepatocytes on denatured collagen in the presence of fibronectin in a manner similar to when Gly-Pro-Ala was added to the medium. These observations lead to the conclusion that plasma membrane dipeptidyl peptidase IV may be involved in the initial phase of fibronectin-mediated cell spreading on collagen.     

Fibronectin-mediated binding and phagocytosis of polystyrene latex beads by baby hamster kidney cells

The binding and phagocytosis of fibronectin (pFN)-coated latex beads by baby hamster kidney (BHK) cells was studied as a function of fibronectin concentration and bead diameter. Cells were incubated with radioactive pFN-coated beads, and total bead binding (cell surface or ingested) was measured as total radioactivity associated with the cells. Of the bound beads, those that also were phagocytosed were distinguished by their insensitivity to release from the cells by trypsin treatment. In continuous incubations, binding of pFN-coated beads to cells occurred at 4 degrees C or 37 degrees C, but phagocytosis was observed only at 37 degrees C. In addition, degradation of 3H-pFN from ingested beads occurred at 37 degrees C, as shown by the release of trichloroacetic acid-soluble radioactivity into the incubation medium. When the fibronectin density on the beads was varied, binding at 4 degrees C and ingestion at 37 degrees C were found to have the same dose-response dependencies, which indicated that pFN densities that permitted bead binding were sufficient for phagocytosis to occur. The fibronectin density for maximal binding of ingestion was approximately 250 ng pFN/cm2. When various sized beads (0.085-1.091 micron), coated with similar densities of pFN, were incubated with cells at 4 degrees C, no variation in binding as a function of bead size was observed. Under these conditions, the absolute amount of pFN ranged from less than 100 molecules on the 0.085-micron beads to greater than 15,000 molecules on the 1.091-micron beads. Based upon these results it can be concluded that the critical parameter controlling fibronectin-mediated binding of latex beads by BHK cells is the spacing of the pFN molecules on the beads. Correspondingly, it can be suggested that the spacing between pFN receptors on the cell surface that is optimal for multivalent interactions to occur is approximately 18 nM. When phagocytosis of various sized beads was compared, it was found that the largest beads were phagocytosed slightly better (two fold) than the smallest beads. This occurred both in continuous incubations of cells with beads and when the beads were prebound to the cells. Finally, the kinetic constants for the binding of 0.085 microM pFN-coated beads to the cells were analyzed. There appeared to be approximately 62,000 binding sites and the KD was 4.03 X 10(-9) M. Assuming a bivalent interaction, it was calculated that BHK cells have approximately 120,000 pFN receptors/cell and the binding affinity between pFN and its receptor is approximately 6 X 10(-5) M.     

Neurite outgrowth promotion by the alternatively spliced region of tenascin-C is influenced by cell-type specific binding.

We have investigated the impact of cellular environment on the neurite outgrowth promoting properties of the alternatively spliced fibronectin type-III region (fnA-D) of tenascin-C. FnA-D promoted neurite outgrowth in vitro when bound to the surface of BHK cells or cerebral cortical astrocytes, but the absolute increase was greater on astrocytes. In addition, different neurite outgrowth promoting sites were revealed within fnA-D bound to the two cellular substrates. FnA-D also promoted neurite outgrowth as a soluble ligand; however, the actions of soluble fnA-D were not affected by cell type. Therefore, we hypothesized that different mechanisms of cellular binding can alter the growth promoting actions of bound fnA-D. We found that fnA-D utilizes two distinct sequences to bind to the BHK cell surface as opposed to the BHK extracellular matrix. In contrast, only one of these sequences is utilized to bind to the astrocyte matrix as opposed to the astrocyte surface. Furthermore, Scatchard analysis indicated two types of receptors for fnA-D on BHK cells and only one type on astrocytes. These results suggest that active sites for neurite outgrowth within fnA-D are differentially revealed depending on cell-specific fnA-D binding sites. Therefore, the function of tenascin-C and its various domains must be considered in terms of cellular context.     

Inhibition of cell adhesion by type V collagen.

Human umbilical vein endothelial cells grew well in dishes coated with collagen types I, II, III, or IV. However, the same cells tended to detach themselves from dishes coated with type V collagen, and cell proliferation in these dishes was inhibited. Such anti-adhesive activity was partially retained by heat-denatured type V collagen or by its alpha 1 chain, but not by its alpha 2 chain. Several other cell types did not adhere to the type V collagen substratum even in the presence of 10% serum. The cell types strongly inhibited from adhering by type V collagen included Swiss mouse 3T3 cells and their MSV-transformants, BALB/c 3T3 cells and their methylcholanthrene-transformants, NIH 3T3 cells and their ras-transformants, BHK cells, CHO-9 cells, CHO-K1 cells, and mouse melanoma B16-F10 cells. Using Swiss mouse 3T3, we studied the effects of type V collagen on cell adhesion to fibronectin in serum-free medium. When the culture dishes were coated with a mixture of fibronectin with various concentrations of type V collagen, the adhesion of the cells was inhibited depending on the concentration of type V collagen. The inhibition of cell adhesion by type V collagen was competitively overcome by increased concentrations of fibronectin. The activity that interferes with the effects of fibronectin was retained mainly by the alpha 1 chain of heat-denatured type V collagen.     

Effects of plant lectins on the adhesive properties of baby hamster kidney cells

We studied the effects of different lectins on the adhesive properties of baby hamster kidney (BHK) cells. The purpose of these studies was to learn more about the cell surface receptors involved in cell adhesion. Three adhesive phenomena were analyzed: 1) the adhesion of BHK cells to lectin-coated substrata; 2) the effects of lectins on the adhesion of cells to substrata coated by plasma fibronectin (pFN); and 3) the effects of lectins on the binding of pFN-coated beads to cells. Initial experiments with fluorescein-conjugated lectins indicated that concanavalin A (Con A), ricinus communis agglutinin I (RCA I), and wheat germ agglutinin (WGA) bound to BHK cells but peanut agglutinin (PNA), soybean agglutinin (SBA), and ulex europaeus agglutinin I (UEA I) dod not bind. All three of the lectins which bound to the cells promoted cell spreading on lectin substrata, and the morphology of the spread cells was similar to that observed with cells spread on pFN substrata. Protease treatment of the cells, however, was found to inhibit cell spreading on pFN substrata or WGA substrata more than on Con A substrata or RCA I substrata. In the experiment of cells with Con A or WGA inhibited cell spreading on pFN substrata, but RCA I treatment had no effect. Finally, treatment of cells with WGA inhibited binding to cells of pFN beads, but neither Con A nor RCA I affected this interaction. These results indicate that the lectins modify cellular adhesion in different ways, probably by interacting with different surface receptors. The possibility that the pFN receptor is a WGA receptor is discussed.     

Polypeptide heterogeneity of hamster and calf fibronectins.

The adhesive glycoprotein fibronectin has been isolated from fresh hamster plasma by affinity chromatography on gelatin coupled to Sepharose beads by the method of Engvall & Ruoslahti [Int. J. Cancer (1979) 20, 1-5]. Polyacrylamide-gel electrophoresis of material heated in sodium dodecyl sulphate and 2-mercaptoethanol shows two prominent polypeptide subunits of approx. mol.wts. 215 000 and 200 000, with variable amounts of lower-molecular-weight fragments. The unexpected polypeptide heterogeneity of different preparations of hamster fibronectins and bovine serum fibronectin is shown to be partly an artefact and is generated during isolation and storage of purified fibronectin. Treatment of each hamster fibronectin subunit or a smaller fragment of approx. mol.wt. 140 000 with thermolysin or trypsin after radioiodination produces similar patterns of tyrpsine-containing peptides, indicating similar primary amino-acid sequences. Antibodies raised against the major subunits of hamster plasma fibronectin were coupled to Sepharose beads and used in conjunction with gelatin affinity chromatography to isolate fibronectins extracted with urea from baby-hamster kidney (BHK) cells and present in the long-term culture medium of these cells. The cell and medium fibronectins are similar to hamster plasma fibronectin in amino-acid and carbohydrate composition and also produce very similar peptide 'maps'. We conclude that the various forms of hamster fibronectins are structurally analogous in agreement with indistinguishable biological properties in mediating the substance adhesion of BKH cells [Pena & Hughes (1978) Cell Biol. Int. Rep. 3, 339-344].     

Isolation and characterization of a novel large protease accumulated in mammalian cells in the presence of inhibitors

We have isolated and characterized a novel, large, multicatalytic protease from mammalian cells. This protease was designated PABI (protease accumulated by inhibitors). When baby hamster kidney (BHK) cells were grown in medium containing leupeptin, a potent serine-cysteine protease inhibitor, the trypsin-like protease activity (PABI) in the cells increased its level more than 100-fold over the control. This increase was also observed in other cultured cells such as COS, HepG2, and skin fibroblast cells. The activity was also elevated by treatment with other protease inhibitors including chymostatin or trans-epoxysuccinyl-L-leucylamide-(4-guanidino)butane. Immunoblot analysis, by employing antisera prepared against the purified PABI, also showed a concomitant increase of this protein in BHK, COS, and HepG2 cells on leupeptin treatment. PABI was purified to a homogeneous state from leupeptin-treated BHK cells. PABI is a glycoprotein of molecular weight 700,000. PABI was found to be a multimer of a major subunit of apparent Mr of 84,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopic analysis. PABI dissociates into subunits only under reducing conditions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PABI has both trypsin-like and chymotrypsin-like protease activities toward synthetic substrates. Both activities were inhibited by phenylmethanesulfonyl fluoride, aprotinin, bovine pancreas trypsin inhibitor, and chymostatin. Leupeptin inhibited only the trypsin-like activity of PABI. p-Chloromercuribenzoate had no effect on either activity. Furthermore, PABI degraded collagen type I and fibronectin. These results indicate that PABI is a novel protease which differs from any known proteases including cytosolic high molecular weight proteases. The physiological function of PABI is yet to be determined.