Adhesion-promoting Activity Research Articles

Inhibition of adhesion-promoting activity of a human salivary protein which promotes adhesion of Streptococcus mutans JBP to hydroxyapatite

Inhibition of adhesion-promoting activity of a human salivary protein which promotes adhesion of Streptococcus mutans JBP to hydroxyapatite

Delineation of a segment of adsorbed salivary acidic proline-rich proteins which promotes adhesion of Streptococcus gordonii to apatitic surfaces

Cells of several strains of Streptococcus gordonii attached in much higher numbers to experimental pellicles formed from samples of submandibular or parotid saliva on hydroxyapatite (HA) beads than to buffer controls. The nature of the salivary components responsible were investigated by preparing experimental pellicles from chromatographic fractions of submandibular saliva obtained from Trisacryl GF 2000M columns. Adhesion of S. gordonii Blackburn was promoted by two groups of fractions. The adhesion-promoting activity in the first group of fractions was associated with the family of acidic proline-rich proteins (PRPs), while that of the second group is as yet unidentified. Experimental pellicles prepared by treating HA with 2 micrograms of pure 150-amino-acid-residue PRPs (PRP-1, PRP-2, and PIF-s) promoted adhesion of S. gordonii Blackburn cells to an extent comparable to that obtained with unfractionated saliva. However, pellicles prepared from a 106-residue PRP (PRP-3) were significantly less effective, and those prepared from the amino-terminal tryptic peptide (residues 1 to 30) of the PRP and the salivary phosphoprotein statherin were completely ineffective in promoting adhesion. Although adhesion of several strains of S. gordonii was promoted by adsorbed PRP-1, the adhesion of several strains of Streptococcus sanguis or Streptococcus oralis was either not affected or only weakly enhanced by this protein. S. gordonii cells bound avidly to PRPs adsorbed onto HA beads, but the streptococci did not appear to bind PRPs in solution, since concentrations of PRP as high as 200 micrograms/ml did not inhibit binding of bacterial cells to pellicles prepared from pure PRP. S. gordonii cells also attached well to PRP or a synthetic decapeptide representing residues 142 to 150 of the PRP when the peptide was linked to agarose beads. Studies with a series of synthetic decapeptides indicated that the minimal segment of PRP which promoted high levels of S. gordonii adhesion was the carboxy-terminal dipeptide Pro-Gln (residues 149 and 150).

Extracellular matrix adhesion-promoting activities of a dermatan sulfate proteoglycan-associated protein (22K) from bovine fetal skin

A 22 x 10(3) Mr protein (abbreviated 22K) that copurifies with dermatan sulfate proteoglycans (DS-PGs) following the biochemical fractionation of bovine fetal skin has been evaluated for adhesion-promoting activity in vitro using Balb/c 3T3 cells, as well as bovine and human dermal fibroblasts. Substrata coated with 22K protein promote attachment of a subset of 3T3 and dermal fibroblasts that respond to plasma fibronectin (pFN) substrata. Cells on 22K protein display partial cytoplasmic spreading, comparable to that of cells adhering to cell-binding fragments of pFN. Adhesion activity of 22K is not due to contamination with known adhesive proteins of dermal matrices and is not dermal cell type-specific, since two classes of neuronal cells also respond effectively to 22K substrata. DS-PGs from cartilage or skin completely inhibit 22K adhesion activity when the PGs are adsorbed to 22K substrata under conditions prohibiting PGs from binding to substrata directly. Cartilage chondroitin/keratan sulfate proteoglycan at much higher concentrations is only partially inhibitory. Inhibition by DS-PGs is mediated by DS chains binding to 22K. Properties of the cell surface 'receptor' for 22K protein were tested by several approaches. It is not cell surface DS-PG, since: (1) cells unable to produce this proteoglycan class also responded; (2) cells treated with chondroitinase ABC responded equally well; and (3) substrata of proteoglycan-binding platelet factor-4 generated responses from cells that were quantitatively and qualitatively different. A synthetic peptide in the medium containing the Arg-Gly-Asp-Ser (RGDS) sequence completely inhibited responses to 22K substrata. This observation, coupled with sequencing data of 22K protein revealing an Arg-Gly-Ala-Thr sequence at residues 151-154, suggest that 22K protein mediates adhesion by cell surface integrin binding. Therefore, this newly discovered matrix protein from skin may serve as a communication link between the dermal fibroblast cell surface and its extracellular matrix environment.

A human salivary protein which promotes adhesion of Streptococcus mutans serotype c strains to hydroxyapatite.

The aim of this study was to investigate the nature of one of the factors in human submandibular-sublingual (SMSL) saliva which promotes the adhesion of Streptococcus mutans serotype c strains to hydroxyapatite (HA) surfaces. Gel filtration chromatography of SMSL saliva on Trisacryl GF2000 gave a void volume peak which contained the major fraction of adhesion-promoting activity for S. mutans JBP to HA. Maximum adhesion-promoting activity, however, eluted slightly later than the maximum 220-nm absorbance of the void volume peak. Gel filtration of the void volume material after treatment with sodium dodecyl sulfate (SDS) gave an early-eluting larger peak followed by a smaller peak with which the adhesion-promoting activity was associated. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed the presence of relatively slowly migrating material associated with the larger inactive peak, presumably mucin, and a faster-migrating band(s) associated with the smaller active peak. SDS-PAGE indicated molecular weights in the range of 300,000 to 350,000 by extrapolation from size standards. Comparison of SMSL from five individuals showed the presence of single bands or double bands associated with adhesion-promoting activity, indicating genetic polymorphism. The active material did not resemble either secretory immunoglobulin A, based on SDS-PAGE and immunoassay, or fibronectin, based on SDS-PAGE, and also differed in molecular weight from salivary mucins and salivary constituents previously reported to promote aggregation of certain oral bacteria, but a relationship to these materials cannot be excluded. This adhesion-promoting material may play a significant role in the initial colonization of tooth surfaces by S. mutans strains.

Adsorbed Salivary Acidic Proline-rich Proteins Contribute to the Adhesion of Streptococcus mutans JBP to Apatitic Surfaces

Experimental pellicles formed on hydroxyapatite (HA) beads from parotid or submandibular saliva promoted the adhesion of Streptococcus mutans JBP cells to a greater extent than did pellicles prepared from buffer, human plasma, or serum. The nature of the salivary components responsible was studied by the preparation of pellicles from fractions of parotid saliva obtained by chromatography on Trisacryl GF 2000 columns. Two groups of fractions promoted attachment of the organism. Components migrating in the high-molecular-weight mucin fraction were most effective, but a later-eluting fraction also possessed adhesion-promoting activity. Subfractionation of the latter material indicated that the adhesion-promoting activity was associated with the acidic proline-rich proteins (PRPs). Pellicles prepared from 10-20-micrograms/mL solutions of pure PRP-1 were effective in promoting attachment of S. mutans JBP cells. PRP-3 was less effective, while human salivary statherin, fibrinogen, fibronectin, type 1 collagen, and the amino-terminal tryptic peptide derived from PRP-1 were ineffective. The quantities of 150-residue and 106-residue PRPs and of statherin, which became incorporated into experimental pellicles prepared from saliva, were estimated with use of radiolabeled protein tracers. The data obtained suggest that these proteins compete for similar binding sites on HA, and that their ratios in saliva would therefore influence the quantity of the larger PRPs that become incorporated into the pellicle. Such competition may contribute to the variability observed in the adhesion-promoting activities of different saliva samples.

Protein-mediated Adhesion of Lactobacillus fermentum Strain 737 to Mouse Stomach Squamous Epithelium

The mechanism of adhesion of Lactobacillus fermentum strain 737 to mouse stomach squamous epithelium was investigated. Adhesion inhibition tests involving chelators, monosaccharides, periodate and concanavalin A and the use of bacteria grown in the presence of tunicamycin failed to clarify the adhesive mechanism. Washed bacterial cells had reduced adhesive capacity, except in the presence of spent broth culture supernatant fraction or cell washings. Spent culture supernatant fractions of erythrosine-supplemented broth did not enhance adhesion of washed cells. The adhesion-promoting factor(s) in the spent broth culture supernatant fractions and cell washings bound to both bacterial and epithelial cell surfaces, but did not promote adhesion of two other Lactobacillus strains which were not of mouse origin, thereby indicating host specificity for the adhesion-promoting activity. Chemical characteristics of the adhesion-promoting factor were determined by pretreatment of the dialysis retentate of spent broth culture supernatant fractions with proteolytic enzymes, concanavalin A-Sepharose or periodate before the adhesion assay. The adhesin was non-dialysable, pronase-sensitive, heat sensitive at 100 degrees C, had no affinity for concanavalin A-Sepharose and contained no carbohydrate groups active in the adhesion process. The protein profiles of dialysis retentates of spent broth culture supernatant fractions after bacterial growth in the absence and presence of erythrosine were determined by 2-dimensional SDS-PAGE. Gel filtration by HPLC was used for purification of an adhesion-promoting fraction. The host-specific adhesion of L. fermentum strain 737 was mediated by a protein, with an Mr of 12-13000, that was not detectable in cells grown in the presence of erythrosine. A model for the mode of binding of the adhesin to host epithelia and bacterial surfaces is proposed.

A novel synthetic peptide from the B1 chain of laminin with heparin-binding and cell adhesion-promoting activities.

Recent studies using solid-phase-binding assays and electron microscopy suggested the presence of a heparin-binding domain between the inner globule of a lateral short arm and the cross region of laminin. Using the information from the amino acid sequence of the B1 chain of laminin, several peptides were synthesized from areas with a low hydropathy index and a high density of lysines and/or arginines. One of these, peptide F-9 (RYVVLPRPVCFEKGMNYTVR), which is derived from the inner globular domain of the lateral short arm, demonstrated specific binding to heparin. This was tested in direct solid-phase binding assays by coating the peptide either on nitrocellulose or on polystyrene and in indirect competition assays where the peptide was in solution and either laminin or heparin was immobilized on a solid support. The binding of [3H]heparin to peptide F-9 was dramatically reduced when heparin but not other glycosaminoglycans other than heparin (dextran sulfate, dermatan sulfate) were used in competition assays. Modification of the free amino groups of peptide F-9 by acetylation abolished its ability to inhibit the binding of [3H]heparin to laminin on polystyrene surfaces. Peptide F-9 promoted the adhesion of various cell lines (melanoma, fibrosarcoma, glioma, pheochromocytoma) and of aortic endothelial cells. Furthermore, when peptide F-9 was present in solution, it inhibited the adhesion of melanoma cells to laminin-coated substrates. These findings suggest that peptide F-9 defines a novel heparin-binding and cell adhesion-promoting site on laminin.

The adult respiratory distress syndrome. Cell populations and soluble mediators in the air spaces of patients at high risk.

In order to better understand the modulation of polymorphonuclear neutrophil influx into the lung during the development of the adult respiratory distress syndrome (ARDS), we evaluated bronchoalveolar lavage fluids from control subjects (n = 9), patients at high risk of developing ARDS (n = 12), and patients with ARDS (n = 11) for cellular and protein content and capacity to promote neutrophil adhesion to tissue culture plastic. Analysis of the lavage fluids from high risk patients and patients with ARDS showed an 8- to 10-fold increase in the total number of cells, an increase in the percentage of neutrophils present (control subjects = 1 +/- 0.4%, high risk = 53 +/- 8%, ARDS = 70 +/- 7%), and a 10- to 40-fold increase in protein content. The adherence of normal neutrophils to plastic surfaces after pretreatment with either concentrated lavage fluid, ultrafiltrates of BALF, or plasma samples was determined to evaluate the neutrophil adherence-promoting activity of each. Lavage fluid from high risk patients and patients with ARDS promoted an approximate 3-fold increase in neutrophil adherence when compared with control lavage fluid. Neutrophil adhesion-promoting activity of the plasma and lavage filtrates (mw less than 500 daltons) was not significantly different from that of control subjects. The adherence-promoting activity found in ARDS lavage was stable at 56 degrees C for 30 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombospondin and the Adhesive Behavior of Platelets

The adhesion-promoting activity of TSP has been examined in four systems. Both the amino-terminal heparin-binding domain and the large globular carboxy-terminal domains have been implicated in the agglutination of erythrocytes and fixed-activated platelets by TSP. Sulfatides specifically bind TSP and may serve as a membrane receptor for the heparin-binding domain of TSP. The large globular domains of TSP have been implicated in secretion-dependent platelet aggregation. Activation-dependent adhesion of platelets to TSP substrates does not appear to be mediated solely by direct interaction of TSP with platelet membrane components, but appears to be mediated in large part by intervening bridging molecules, such as fibronectin or fibrinogen. Thus, different regions of the complex TSP molecule are of varying importance in the different systems. No single simple system appears adequate for exploration of the adhesion-promoting activity of TSP.

Thrombospondin Promotes Cell-Substratum Adhesion

The physiological role of the platelet-secreted protein thrombospondin (TSP) is poorly understood, although it has been postulated to be involved in platelet aggregation and cellular adhesion. In this report, TSP isolated from human platelets was found to promote, in vitro, the cell-substratum adhesion of a variety of cells, including platelets, melanoma cells, muscle cells, endothelial cells, fibroblasts, and epithelial cells. The adhesion-promoting activity of TSP was species independent, specific, and not due to contamination by fibronectin, vitronectin, laminin, or platelet factor 4. The cell surface receptor for TSP is protein in nature and appears distinct from that for fibronectin.

Identification of two distinct regions of the type III connecting segment of human plasma fibronectin that promote cell type-specific adhesion.

The principal region of the human plasma fibronectin molecule mediating the adhesion of melanoma cells appears to be the alternatively spliced type III connecting segment (IIICS (Humphries, M. J., Akiyama, S. K., Komoriya, A., Olden, K., and Yamada, K. M. (1986a) J. Cell Biol., in press]. A series of overlapping synthetic peptides spanning the entire IIICS (CS peptides) were examined for their effects on B16-F10 melanoma cell adhesion to the parent fibronectin molecule. Two nonadjacent CS peptides, designated CS1 and CS5, were inhibitory. In contrast, neither inhibited fibronectin-mediated spreading of fibroblastic baby hamster kidney cells. When N-terminal cysteine derivatives of the CS peptides were conjugated to IgG by covalent cross-linking with N-succinimidyl-3(2-pyridyldithio)propionate, both the CS1 and CS5 conjugates promoted B16-F10 melanoma cell spreading. All conjugates were inactive for spreading of baby hamster kidney cells, confirming the cell type specificity of the IIICS adhesion site. Determination of the amounts of CS peptide required to support melanoma cell adhesion revealed that the activity of CS1 was only 2.4-fold lower than that of the intact fibronectin molecule. CS5 was approximately 320-fold less active than fibronectin, suggesting that the CS1 region may be the major site of interaction with the melanoma cell surface. The adhesion-promoting activities of CS1-IgG and CS5-IgG were additive as were the inhibitory activities of the free peptides for B16-F10 cell spreading on fibronectin. These findings suggest that both regions of the IIICS can function separately or together in mediating the interaction of melanoma cells with fibronectin. Since CS1 and CS5 are each found in separate alternatively spliced regions of the IIICS, it is conceivable that the adhesion-promoting activity of fibronectin for different cell types may be under complex regulation.

Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion.

We have compared the molecular specificities of the adhesive interactions of melanoma and fibroblastic cells with fibronectin. Several striking differences were found in the sensitivity of the two cell types to inhibition by a series of synthetic peptides modeled on the Arg-Gly-Asp-Ser (RGDS) tetrapeptide adhesion signal. Further evidence for differences between the melanoma and fibroblastic cell adhesion systems was obtained by examining adhesion to proteolytic fragments of fibronectin. Fibroblastic BHK cells spread readily on fl3, a 75-kD fragment representing the RGDS-containing, "cell-binding" domain of fibronectin, but B16-F10 melanoma cells could not. The melanoma cells were able to spread instead on f9, a 113-kD fragment derived from the large subunit of fibronectin that contains at least part of the type III connecting segment difference region (or "V" region); f7, a fragment from the small fibronectin subunit that lacks this alternatively spliced polypeptide was inactive. Monoclonal antibody and fl3 inhibition experiments confirmed the inability of the melanoma cells to use the RGDS sequence; neither molecule affected melanoma cell spreading, but both completely abrogated fibroblast adhesion. By systematic analysis of a series of six overlapping synthetic peptides spanning the entire type III connecting segment, a novel attachment site was identified in a peptide near the COOH-terminus of this region. The tetrapeptide sequence Arg-Glu-Asp-Val (REDV), which is somewhat related to RGDS, was present in this peptide in a highly hydrophilic region of the type III connecting segment. REDV appeared to be functionally important, since this synthetic tetrapeptide was inhibitory for melanoma cell adhesion to fibronectin but was inactive for fibroblastic cell adhesion. REDV therefore represents a novel adhesive recognition signal in fibronectin that possesses cell type specificity. These results suggest that, for some cell types, regulation of the adhesion-promoting activity of fibronectin may occur by alternative mRNA splicing.

Human fibronectin contains distinct adhesion- and motility-promoting domains for metastatic melanoma cells.

The active migration of tumor cells through extracellular matrices has been proposed to play a role in certain aspects of metastasis. Metastatic tumor cells migrate in vitro in response to substratum-bound adhesive glycoproteins such as fibronectin. The present studies use affinity-purified proteolytic fragments of fibronectin to determine the nature of adhesion- and/or motility-promoting domains within the protein. Two distinct fragments were identified with cell adhesion-promoting activities. By a number of criteria, the adhesive activity promoted by these two fragments was distinct. One fragment, a 75-kD tryptic fragment purified by monoclonal antibody chromatography, promoted the adhesion, spreading, and haptotactic motility of melanoma cells. Experiments using a synthetic cell attachment peptide in solution indicated that at least part of the attachment activity exhibited by the 75-kD fragment is mediated by the sequence arg-gly-asp-ser. It was not possible to demonstrate migration-stimulating activity using a small (11.5 kD) peptic fragment containing this sequence (Pierschbacher, M.D., E. G. Hayman, and E. Ruoslahti, 1981, Cell, 26:259-267) suggesting that another cell-binding activity within the 75 kD fragment distinct from arg-gly-asp-ser might be required for motility. The second fragment that stimulated melanoma adhesion was a 33-kD tryptic/catheptic carboxyl-terminal heparin-binding fragment, which is localized to the A chain of fibronectin. This fragment promotes adhesion and spreading but not the motility of these cells. Melanoma adhesion to this heparin-binding fragment was sensitive to the effects of cycloheximide, which contrasted adhesion to the haptotaxis-promoting fragment. Importantly, these studies illustrate that haptotaxis in response to fibronectin is not due to simple adhesion gradients of this protein. The results are discussed in light of a model for multiple distinct cell surface constituents mediating cell adhesion and motility on fibronectin.

Increased adhesion response of anaplastic glioma cells to nerve growth factor and the presence of specific receptors.

A trypsin-degradable nerve growth factor (NGF) receptor associated with the phospholipid component of the surface membrane has been detected on F98 anaplastic glioma cells. NGF also bound to the nucleus of F98 cells. Bound NGF was not displaceable by insulin, cytochrome C, growth hormone, or bovine serum albumin. Specific binding of NGF occurred with a Kd of 8.79 X 10(-12) M as determined by Scatchard analysis with approximately 34,000 receptors per cell. Specific NGF binding was also evident to C6 rat glioma cells and IMR-32 human neuroblastoma cells, but not to 3T3 mouse fibroblasts. These observations coupled with previous findings suggest that the NGF receptor may be a marker found on cells of neural derivation. As little as 1 ng/ml NGF caused an increase in the adhesiveness of F98 cells to culture flasks. Increased adhesiveness could be observed in as little as 5 min and was apparent for at least 45 min. At 25 min in NGF-containing medium, 24 +/- 3% of the cells adhered to the flasks compared to 13 +/- 1% of control cells. The NGF-induced increase in adhesiveness was not duplicated by epidermal growth factor, insulin, cytochrome c, bovine serum albumin, dibutyryl cyclic AMP, or sodium butyrate. Oxidized NGF blocked the effect of native NGF, but had little or no adhesion-promoting activity itself. Pretreatment of the cells with NGF was also effective in promoting adhesion, even though nerve growth factor was not added to the binding medium. The effect of this pretreatment was reversible; when NGF-pretreated cells were grown in medium without supplemental NGF, the adhesiveness of the cells returned to control levels or lower.

Glycosaminoglycans that bind cold-insoluble globulin in cell-substratum adhesion sites of murine fibroblasts.

Glycosaminoglycans (GAGs) and glycoprotein-derived glycopeptide from mouse BALB/c3T3 and simian virus 40-transformed 3T3 whole cells or their adhesion sites, which are left bound to the serum-coated tissue culture substratum after detachment of cells mediated by [ethylenebis-(oxyethylenenitrilo]tetraacetic acid (EGTA), were analyzed for specific binding to Sepharose columns derivatized with cold-insoluble globulin (CIg). CIg is the serum-contained form of fibronectin and is required for the adhesion of these fibroblasts to the substratum. Of the various GAGs present in these fractions of either cell type, only the highly N-sulfated sequences of heparan sulfate and a small subset of dermatan sulfate bind to CIg-Sepharose. There was no detectable binding of glycopeptide, undersulfated heparan sulfate, the various chondroitin species, or hyaluronate. Adhesion sites from newly attaching cells were greatly enriched in CIg-binding heparan sulfate when compared to long-term-growth adhesion sites or EGTA-detached cells. Various properties of binding were determined. The reference standard standard GAGs heparin (or heparan sulfate) and dermatan sulfate were able to displace bound radiolabeled adhesion site GAG from the column, whereas the other GAGs had no effect. CIg has been shown to be the only adhesion-promoting activity in the serum layer of this culture system. Because these fibroblast adhesion sites do not contain collagen, which could potentially mediate adhesion to the substratum-bound CIg, these data support other evidence that multivalent heparan sulfate proteoglycans mediate substratum adhesion of these cells by coordinate binding to fibronectin on the cell surface and CIg on the substratum.

Cell to substratum adhesion-promoting activity released by normal and virus-transformed cells in culture.

It is demonstrated here that cultured fibroblasts release into their medium a nondialyzable, protease-sensitive factor(s) capable of promoting the adhesion and spreading of virus-transformed rat fibroblasts on a plastic substratum. A relatively sensitive biological assay is described for quantitation of the adhesion-promoting factor (APF) activity in serum-free, conditioned medium harvested from the cultures. Evidence is presented which indicates that the primary mode of action of the APF is by binding to and modifying the properties of the substratum. Conditioned media harvested after 24 h of incubation in similarly populated cultures of normal fibroblasts of diverse animal species exhibited similar levels of APF activity. However, conditioned media obtained from Rous sarcoma virus (Prague strain)-transformed and avian sarcoma virus B77-transformed rat fibroblasts exhibited three- to sixfold lower levels of APF activity than media conditioned in parallel cultures of heterologous or homologous normal fibroblasts. Cultivation of B77 virus-transformed rat cells in the presence of dibutyryl cyclic AMP and theophylline led to as much as a sevenfold increase in the level of APF activity appearing in the culture medium, with a concomitant increase in the adhesiveness of the cells to the culture substratum. The results support the role of extracellular macromolecules in cell to substratum adhesion. It is postulated that the reduced adhesiveness of transformed cells to a substratum may be at least partially owing to a deficiency in the production and/or release of APF-like macromolecules.