A cell attachment peptide from human C-reactive protein.

The serum acute phase reactant, C-reactive protein (CRP), is selectively deposited at sites of tissue damage and degraded by neutrophils into biologically active peptides. A synthetic peptide corresponding to residues 27-38 present in each of the five identical subunits of CRP mediated cell attachment activity in vitro. Although the CRP-derived peptide contains a Tuftsin (TKPR)-like sequence at its amino-terminus, the Tuftsin tetrapeptide itself, as well as several synthetic peptides of CRP, failed to inhibit the cell-attachment activity to the CRP-derived peptide. Peptides containing the sequences responsible for the cell attachment activity of the extracellular matrix proteins, fibronectin (Fn) and laminin, failed to inhibit the CRP-derived peptide cell attachment activity. However, the addition of the RGDS and RGDSPASSLP cell-binding peptides of Fn to cells enhanced attachment to the active peptide from CRP. In the converse experiment, the cell-binding peptide of CRP did not influence cell attachment to Fn or laminin. A peptide corresponding to the same stretch of amino acid residues within the homologous Pentraxin, serum amyloid P-component (SAP), displayed nearly identical cell-attachment activity. Several monoclonal antibodies (mAb) specific for the CRP-derived cell-binding peptide neutralized its cell-attachment activity. These mAbs reacted with intact CRP and neutralized the cell-binding activity of CRP itself. The findings suggest that a peptide with cell-binding activity could be generated from the breakdown of CRP and then contribute directly to cellular events leading to tissue repair.     

Synthetic pentapeptide from the B1 chain of laminin promotes B16F10 melanoma cell migration

Laminin is a basement membrane-specific glycoprotein that promotes cell adhesion, proliferation, differentiation, and tumor cell migration. Synthetic peptides from the amino acid sequence deduced from a cDNA clone of the B1 chain of laminin were tested for their ability to promote the migration of B16F10 melanoma cells. A peptide, CDPGYIGSR, that is able to mediate epithelial cell attachment to laminin was found to promote migration, and the constituent pentapeptide YIGSR was also active but to a lesser degree. This nine-amino acid peptide blocked migration of melanoma cells to laminin but had no effect on migration to fibronectin. These data suggest that the cell-binding site and migration site on laminin share a common sequence that is unique to laminin.     

Cell attachment peptide of C-reactive protein: critical amino acids and minimum length

Human C-reactive protein (CRP) is an acute phase blood component that accumulates at sites of tissue damage and necrosis and is degraded by neutrophils to biologically active peptides. A dodecapeptide composed of amino acids 27–38 of CRP mediates cell attachment in vitro. This peptide was designated the cell-binding peptide (CB-Pep) of CRP. Characterization of the interaction between fibroblasts and modified synthetic peptides with sequential deletions from either the N-terminus or C-terminus revealed that the minimal sequence for cell attachment or inhibition of cell attachment to the CB-Pep was Phe-Thr-Val-Cys-Leu , which corresponds to residues 33–37 within each of the five 206 amino acid subunits of CRP. The pentapeptide by itself mediated cell attachment. Substitutions for each residue within the CB-Pep indicated that the critical residues for activity were Phe-33 and Thr-34. This cell-binding pentapeptide represents a recognition motif for cell adhesion not found in other proteins.     

Rat mast cell carboxypeptidase: amino acid sequence and evidence of enzyme activity within mast cell granules

The amino acid sequence of rat mast cell carboxypeptidase has been determined. The major form has 308 residues; a minor form has an additional (glutamyl) residue at the amino terminus that may indicate an alternate cleavage site during zymogen activation. The enzyme is homologous to pancreatic carboxypeptidases A and B, with conservation of the functional amino acid residues of the active site. The putative substrate binding site resembles that of carboxypeptidase A, although other structural features bear more similarity to carboxypeptidase B. Mast cell carboxypeptidase retains enzymatic activity toward a peptide substrate (angiotensin I) while bound within the granular matrix of the rat connective tissue mast cells. Evidence is presented to suggest that a cluster of positively charged lysyl and arginyl residues binds the enzyme to the negatively charged heparin of the granular matrix but leaves the active site exposed to bind and cleave peptide substrates.     

Multiple cell surface receptors for the short arms of laminin: alpha 1 beta 1 integrin and RGD-dependent proteins mediate cell attachment only to domains III in murine tumor laminin

Cell surface molecules that interact with the cross formed by the three short arms of murine tumor laminin were studied using thermal perturbation, antibody and peptide blocking, and affinity chromatography. Several potential receptors for the laminin short arms were revealed that differed from those mediating cell attachment to the E8 (long arm) fragment. Two cell lines, Rugli and L8 attached well to E1-X (short arm) fragments of laminin. This attachment was blocked by antibodies against alpha 1 integrin chains. Other cells were unable to attach strongly to E1-X, but attached to P1. This attachment was unaffected by anti-beta 1 integrin antibodies, but specifically blocked by the peptide GRGDS. By contrast, binding of Rugli cells was RGD independent and blocked by anti-beta 1 integrin antibodies. G7 and C2C12 myoblasts were very sensitive to GRGDS (ID50 approximately 2 micrograms.ml-1) for attachment to P1 which implied that a non-beta 1 series integrin, possibly alpha v beta 3, was involved. On heat denaturation of P1(3) attachment remained sensitive to RGDS and ID50 was unchanged. On heat denaturation of E1-X, attachment remained sensitive to RGDS but the ID50 increased to approximately 200 micrograms.ml-1. Cellular beta 1 integrins were retained on laminin affinity columns. A beta 1 integrin with an approximately 190 kD alpha-chain could be isolated from Rugli cells whose attachment could be blocked by anti-alpha 1 antibodies and not from cells blocked by RGDS peptides. Anti-alpha 1 antibodies blocked Rugli attachment to native laminin, but only when the E8 cell binding sites on laminin were also blocked. Thus, a receptor related to alpha 1 beta 1 integrin can function simultaneously with a receptor for E8. Anti-alpha 1 also blocked attachment to heated laminin, suggesting that the heat-stable attachment activity in laminin involved the E1-X binding site. Thus, at least two putative receptors mediate attachment to the short arms of laminin. One, related to alpha 1 beta 1 integrin, recognizes RGDS-independent sites in E1-X defined by P1 (within domains III, IIIa, IIIb), and one is an RGD-dependent molecule recognizing sites in P1, and is not a beta 1 integrin.     

Spacer-mediated display of active lipase on the yeast cell surface

We have constructed a Saccharomyces cerevisiae strain displaying an active lipase on the cell surface by cell surface engineering. The gene encoding Rhizopus oryzae lipase (ROL) was fused with the genes encoding the pre-alpha-factor leader sequence and the C-terminal half of alpha-agglutinin including the glycosylphosphatidylinositol-anchor attachment signal. The constructed gene was overexpressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. Linker peptides (spacers) consisting of the Gly/Ser repeat sequence were inserted at the C-terminal portion of ROL to enhance lipase activity by preserving the conformation of the active site near the C-terminal portion. Localization of the expressed ROL on the cell surface was confirmed by immunofluorescence microscopy. The ROL displayed on the yeast cell wall exhibited activity toward soluble 2,3-dimercaptopropan-1-ol tributyl ester (BALB) and insoluble triolein. The insertion of linker peptides effected the activity towards BALB, thereby demonstrating that the optimal length of linker peptides was present. The activity towards triolein was higher in lipases with longer linker peptides. ROL displayed on the cell wall exhibited a comparable and/or higher activity towards triolein than the secreted form of the enzyme. This is the first report of an active lipase displayed on the cell surface. Furthermore, insertion of a linker peptide of the appropriate length as a spacer may be an improved method to effectively display enzymes, especially those having the active region at the C-terminal portion, on the cell surface.     

Cartilage oligomeric matrix protein promotes cell attachment via two independent mechanisms involving CD47 and αVβ3 integrin

Cartilage oligomeric matrix protein (COMP) is a pentameric ~524 kDa multidomain extracellular matrix protein and is the fifth member of the thrombospondin family. COMP is abundantly expressed in proliferating and hypertrophic chondrocytes of the growth plate, articular cartilage, synovium, tendon, and ligament. The spatial localization of COMP highlights its importance in the phenotypes of pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED), COMP disorders that are characterized by disproportionate short stature, brachydactyly, scoliosis, early-onset osteoarthritis, and joint hypermobility. In this study, the role of COMP in ligament was investigated with a series of cell attachment assays using ligament cells binding to COMP. A dose-dependent cell attachment activity was found, which was inhibited by a peptide containing the SFYVVMWK amino acid sequence derived from the globular C-terminal domain of COMP. This activity was independent of the recently described RGD-dependent attachment activity. Function-blocking antibodies to CD47 and αVβ3 integrin reduced cell attachment to COMP, implicating the participation of these cell surface molecules in COMP cell binding. Immunofluorescence studies showed that cell attachment to COMP induced the formation of lamellae containing F-actin microspikes associated with fascin. We propose that COMP promotes cell attachment via two independent mechanisms involving cell surface CD47 and αVβ3 integrin and that a consequence of cell attachment to COMP is the specific induction of fascin-stabilized actin microspikes.     

Analysis of tissue transglutaminase function in the migration of Swiss 3T3 fibroblasts: the active-state conformation of the enzyme does not affect cell motility but is important for its secretion

Increasing evidence suggests that tissue transglutaminase (tTGase; type II) is externalized from cells, where it may play a key role in cell attachment and spreading and in the stabilization of the extracellular matrix (ECM) through protein cross-linking. However, the relationship between these different functions and the enzyme's mechanism of secretion is not fully understood. We have investigated the role of tTGase in cell migration using two stably transfected fibroblast cell lines in which expression of tTGase in its active and inactive (C277S mutant) states is inducible through the tetracycline-regulated system. Cells overexpressing both forms of tTGase showed increased cell attachment and decreased cell migration on fibronectin. Both forms of the enzyme could be detected on the cell surface, but only the clone overexpressing catalytically active tTGase deposited the enzyme into the ECM and cell growth medium. Cells overexpressing the inactive form of tTGase did not deposit the enzyme into the ECM or secrete it into the cell culture medium. Similar results were obtained when cells were transfected with tTGase mutated at Tyr(274) (Y274A), the proposed site for the cis,trans peptide bond, suggesting that tTGase activity and/or its tertiary conformation dependent on this bond may be essential for its externalization mechanism. These results indicate that tTGase regulates cell motility as a novel cell-surface adhesion protein rather than as a matrix-cross-linking enzyme. They also provide further important insights into the mechanism of externalization of the enzyme into the extracellular matrix.     

Identification of an amino acid sequence in laminin mediating cell attachment, chemotaxis, and receptor binding

We have probed for active sites in the B1 chain of laminin using synthetic peptides comprising certain regions of its amino acid sequence as deduced from cDNA clones. An antibody to a 19-mer from domain III inhibited attachment of HT-1080 and CHO cells to laminin, while the peptide itself was inactive. A nearby peptide (CDPGYIGSR) from domain III with homology to epidermal growth factor was synthesized and found to be one of the principle sites in laminin mediating cell attachment, migration, and receptor binding.     

The lytic enzyme of the Pseudomonas phage phi 6. Purification and biochemical characterization.

The lytic enzyme of the lipid-containing bacteriophage phi 6, protein P5, has been purified to apparent homogeneity from disrupted viral particles. The enzyme is a monomer with a molecular mass of approx. 24 kDa. The optimal pH for P5 activity is 8.5 and the protein is readily inactivated at temperatures above 20 degrees C. Protein P5 is active against several Gram-negative bacteria, but no activity against Gram-positive species was detected. Analysis of cell wall digests indicates that P5 is not a glycosidase, but an endopeptidase splitting the peptide bridge formed by meso-diaminopimelic acid and D-alanine.     

A soluble enzyme activity that attaches free diaminopimelic acid to bdelloplast peptidoglycan

An enzyme activity, responsible for the attachment of diaminopimelic acid (DAP) to bdelloplast wall peptidoglycan, was studied in an in vitro, cell-free system. Most of the activity was found in the high-speed (20000g) supernatant fraction of homogenates of bdelloplasts prepared from a culture of the intracellular bacterium Bdellovibrio bacteriovorus 109J, growing synchronously within cells of Escherichia coli. Peptidoglycan preparations obtained either from E. coli ML35 or from the walls of bdelloplasts synchronously cultured for 40 or 90 min served as the acceptors in this reaction, whereas cell wall or peptidoglycan preparations obtained from Gram-positive bacteria could not function as acceptors of DAP. The attachment activity had an apparent Km value for DAP of 10 microM; for bdelloplast peptidoglycan, it was approximately 0.43 mg/mL, which is 13 microM with respect to peptidoglycan disaccharide peptide units. DAP attachment was partially inhibited by the structural analogues lanthionine, L-ornithine, beta-aminobutyric acid, and D-serine, as well as the cell wall synthesis inhibitors penicillin G, ampicillin, and cephalexin. This enzyme activity is present only during the intracellular stage of the bdellovibrio's developmental growth cycle and may serve a stage-specific function of biochemically modifying the cell in which it grows.     

Biologically active synthetic peptides as probes of embryonic development: a competitive peptide inhibitor of fibronectin function inhibits gastrulation in amphibian embryos and neural crest cell migration in avian embryos

We describe a new method for analyzing embryonic events dependent on a specific peptide recognition signal. A short, specific amino acid sequence in fibronectin has been implicated as a recognition site in fibronectin-mediated interactions. Fibroblast adhesion to fibronectin is competitively inhibited by certain synthetic peptides, including the decapeptide Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro, which appears to contain the cell recognition sequence. We found that this peptide inhibited both amphibian gastrulation and avian neural crest cell migration in vivo, as well as the attachment and migration of neural crest cells in vitro. These processes are major cell migratory events previously suggested to involve fibronectin. Negative controls included another conserved fibronectin peptide from the collagen-binding region containing the sequence Cys-Gln-Asp-Ser-Glu-Thr-Arg-Thr-Phe-Tyr and another peptide. Our results demonstrate the feasibility of using synthetic peptides directed at recognition sites in extracellular proteins as probes of morphogenetic processes, and they provide further support for the hypothesis that fibronectin is involved in gastrulation and neural crest cell migration.     

A novel peptide from amyloid P component supports cell attachment.

Amyloid P component is a glycoprotein found in association with connective tissues throughout the body and is also a component of human serum. We have identified a dodecapeptide from amyloid P component which is capable of supporting the attachment of a wide variety of cells to the surface of polystyrene plastic dishes. 83% of the activity is confined to a hexapeptide, FTLCFR. Saturation of cell attachment occurs at a peptide concentration of 100 micrograms/ml used to coat the plastic. These results indicate that the active peptide may represent a functional property of amyloid P component which heretofore has no function.     

Mechanism of interference mediated by human parainfluenza virus type 3 infection

Viral interference is characterized by the resistance of infected cells to infection by a challenge virus. Mechanisms of viral interference have not been characterized for human parainfluenza virus type 3 (HPF3), and the possible role of the neuraminidase (receptor-destroying) enzyme of the hemagglutinin-neuraminidase (HN) glycoprotein has not been assessed. To determine whether continual HN expression results in depletion of the viral receptors and thus prevents entry and cell fusion, we tested whether cells expressing wild-type HPF3 HN are resistant to viral infection. Stable expression of wild-type HN-green fluorescent protein (GFP) on cell membranes in different amounts allowed us to establish a correlation between the level of HN expression, the level of neuraminidase activity, and the level of protection from HPF3 infection. Cells with the highest levels of HN expression and neuraminidase activity on the cell surface were most resistant to infection by HPF3. To determine whether this resistance is attributable to the viral neuraminidase, we used a cloned variant HPF3 HN that has two amino acid alterations in HN leading to the loss of detectable neuraminidase activity. Cells expressing the neuraminidase-deficient variant HN-GFP were not protected from infection, despite expressing HN on their surface at levels even higher than the wild-type cell clones. Our results demonstrate that the HPF3 HN-mediated interference effect can be attributed to the presence of an active neuraminidase enzyme activity and provide the first definitive evidence that the mechanism for attachment interference by a paramyxovirus is attributable to the viral neuraminidase.     

Use of a solid-phase photoaffinity reagent to label a steroid binding site: application to the delta 5-3-ketosteroid isomerase of Pseudomonas testosteroni

In order to extend our analysis of the reactions that occur during the active site directed photoinactivation of delta 5-3-ketosteroid isomerase sensitized by unsaturated steroid ketone photoaffinity reagents, the site of covalent attachment has been identified. A solid-phase photoaffinity reagent, delta 6-testosterone-agarose, has been employed for this purpose; this type of reagent, in contrast to solution-phase reagents, facilitated the recovery of a peptide fragment of the isomerase bearing the residue at which covalent attachment had occurred. Amino acid analysis and sequence determination of the peptide provided evidence that the site of attachment was aspartate-38. This result, in combination with the low-resolution crystallographic structure of the enzyme [Westbrook, E. M., Piro, O. E., & Sigler, P. B. (1984) J. Biol. Chem. 259, 9096-9103], suggests that aspartate-38 is located in the vicinity of the bottom of the steroid-binding pit. The potential usefulness of solid-phase photoaffinity reagents in the identification of sites of covalent attachment on target proteins such as hormone receptors is discussed.     

A recombinant, arginine-glycine-aspartic acid (RGD) motif from foot-and-mouth disease virus binds mammalian cells through vitronectin and, to a lower extent, fibronectin receptors

The cell-binding abilities of a recombinant, RGD-containing peptide from foot-and-mouth disease virus (FMDV) have been characterized in HeLa and BHK cells. This peptide represents the aa sequence of the solvent-exposed G-H loop of protein VP1 which is involved in cell recognition and infection. The efficiency of the viral motif in promoting cell attachment and spreading is comparable to that shown by fibronectin or vitronectin. Cell binding is inhibited by a monoclonal antibody directed against a viral, RGD-involving B-cell epitope and also by sera against vitronectin (_Vβ₃/β₅) and fibronectin (₅β₁) receptors. In addition, a synthetic RGD peptide, which is a ligand for both integrins, prevents the cell binding mediated by the FMDV domain. These data demonstrate that the FMDV RGD motif is a potent ligand for cell-receptor integrins and sufficient to promote cell attachment to susceptible cells mainly through the vitronectin receptor.     

Inhibition of in vitro tumor cell invasion by Arg-Gly-Asp-containing synthetic peptides [published erratum appears in J Cell Biol 1989 Jun;108(6):following 2546]

The interaction of cells with extracellular matrix components such as fibronectin, vitronectin, and type I collagen has been shown to be mediated through a family of cell-surface receptors that specifically recognize an arginine-glycine-aspartic acid (RGD) amino acid sequence within each protein. Synthetic peptides containing the RGD sequence can inhibit these receptor-ligand interactions. Here, we use novel RGD-containing synthetic peptides with different inhibition properties to investigate the role of the various RGD receptors in tumor cell invasion. The RGD-containing peptides used include peptides that inhibit the attachment of cells to fibronectin and vitronectin, a peptide that inhibits attachment to fibronectin but not to vitronectin, a cyclic peptide with the opposite specificity, and a peptide, GRGDTP, that inhibits attachment to type I collagen in addition to inhibiting attachment to fibronectin and vitronectin. The penetration of two human melanoma cell lines and a glioblastoma cell line through the human amniotic basement membrane and its underlying stroma was inhibited by all of the RGD-containing peptides except for the one that inhibits only the vitronectin attachment. Various control peptides lacking RGD showed essentially no inhibition. This inhibitory effect on cell invasion was dose-dependent and nontoxic. A hexapeptide, GRGDTP, that inhibits the attachment of cells to type I collagen in addition to inhibiting fibronectin- and vitronectin-mediated attachment was more inhibitory than those RGD peptides that inhibit only fibronectin and vitronectin attachment. Analysis of the location of these cells that were prevented from invading indicated that they attached to the amniotic basement membrane but did not proceed further into the tissue. These results suggest that interactions between RGD-containing extracellular matrix adhesion proteins and cells are necessary for cell invasion through tissues and that fibronectin and type I collagen are important for this process.     

Structure of the cell wall of Bacillus stearothermophiluys: mode of action of a thermophilic bacteriophage lytic enzyme

The mode of action of a bacteriophage lytic enzyme on cell walls of Bacillus stearothermophilus (NCA 1503-4R) has been investigated. The enzyme is an endopeptidase which catalyzes the hydrolysis of the l-alanyl-d-glutamyl linkage in peptide subunits of the cell wall peptidoglycan. Preliminary studies on the soluble components in lytic cell wall digests indicate that the glycan moiety is composed of alternating glucosamine and muramic acid; one half of the muramic acid residues contain the tripeptide, l-alanyl-d-glutamyldiaminopimelic acid, and the remaining residues contain the tetrapeptide, l-alanyl-d-glutamyldiaminopimeyl-d-alanine. Almost one half of the peptide subunits are involved in cross-linkages of chemotype I. A structure for the cell wall peptidoglycan is proposed in the light of these findings.     

Enzyme histochemistry of rat mast cell tryptase

Fixation and staining conditions for rat mast cell tryptase and its histochemical distribution in different rat tissues were investigated. Prostate, skin, lung, gut, stomach and salivary glands were fixed in either aldehyde or Carnoy fixatives and then frozen or embedded in paraffin wax. Preservation of tryptase enzymic activity against peptide substrates required aldehyde fixation and frozen sectioning. Of the peptide substrates examined, z-Ala-Ala-Lys-4-methoxy-2-naphthylamide and z-Gly-Pro-Arg-4-methoxy-2-naphthylamide proved the most effective for the demonstration of tryptase. Double staining by enzyme cytochemistry followed by immunological detection of tryptase showed that, in all tryptase-containing mast cells, the enzyme is at least in part active. Conventional dye-binding histochemistry was used to confirm the identity of mast cells. Aldehyde-fixed mucosal mast cells required a much shorter staining time with Toluidine Blue if tissue sections were washed directly in t-butyl alcohol. Double staining by enzyme cytochemistry and dye binding showed that tryptase is absent from mucosal and subepidermal mast cells, which are also smaller in size and appear to contain fewer granules than connective tissue mast cells. This study demonstrates that rat mast cell tryptase, unlike tryptases in other species, is a soluble enzyme. It is stored in an active form and is absent from some mast cell subpopulations in mucosa, skin and lung. © 1998 Chapman & Hall