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PubMed2004

Characterization of laminin 5B and NH2-terminal proteolytic fragment of its alpha3B chain: promotion of cellular adhesion, migration, and proliferation.

Various laminin isoforms have specific biological functions depending on their structures. Laminin 5A, which consists of the three truncated chains alpha3A, beta3, and gamma2, is known to have strong activity to promote cell adhesion and migration, whereas a laminin 5 variant consisting of a full-sized alpha3 chain (alpha3Beta) and the beta3 and gamma2 chains, laminin 5B, has not been characterized yet. In the present study, we for the first time cloned a full-length human laminin alpha3B cDNA and isolated the human laminin 5B protein. The molecular size of the mature alpha3B chain (335 kDa) was approximately twice as large as the mature alpha3A chain in laminin 5A. Laminin 5B had significantly higher cell adhesion and cell migration activities than laminin 5A. In addition, laminin 5B potently stimulated cell proliferation when added into the culture medium directly. Furthermore, we found that the alpha3B chain undergoes proteolytic cleavage releasing a 190-kDa NH(2)-terminal fragment. The 190-kDa fragment had activities to promote cellular adhesion, migration, and proliferation through its interaction with integrin alpha(3)beta(1). These activities of the NH(2)-terminal structure of the alpha3B chain seem to contribute to the prominent biological activities and the physiological functions of laminin 5B.

Cell Adhesion↗

PubMed1993

Neural crest cell interaction with type VI collagen is mediated by multiple cooperative binding sites within triple-helix and globular domains.

Collagen type VI (Col VI) is a primary constituent of the extracellular matrix encountered by migrating avian neural crest cells in situ and is effective in promoting attachment and motility of these cells in vitro. In this study, we have explored the molecular mechanisms of neural crest-Col VI interaction by using quantitative assays for cell attachment and migration in vitro, proteolytic fragments of the collagen, and a panel of domain-specific monoclonal antibodies. Removal of the predominant portion of the amino-terminal globular domains of Col VI tetramers by pepsin digestion (P6 fragment) resulted in a > fivefold decrease in their cell adhesion and motility-promoting activity. Further digestion of P6 with bacterial collagenase, which causes a complete loss of the amino-terminal domains plus an adjacent triple-helical segment, did not affect adhesion but reduced migration down to 40% of that seen on undigested P6. Untreated and pepsin-digested Col VI monomers were significantly less effective than their tetrameric counterparts and a M(r) 200,000 fragment, generated from pepsin-digested monomers by a second pepsin treatment, only retained 40% of the motility-promoting activity while preserving the adhesive capacity. A mixture of amino- and carboxyl-terminal globular domains supported both cell attachment and migration. While neural crest cells adhered equally well to the individual intact alpha 1 (VI)/alpha 2(VI) and alpha 3(VI) chains, they migrated most extensively on the alpha 3(VI) chain. Conversely, pepsin-digested individual alpha chains were significantly less effective in promoting cell adhesion and locomotion. Selective preincubation of Col VI microfilaments and isolated tetramers with a panel of monoclonal antibodies against triple helix, carboxyl-terminal, and amino-terminal epitopes of the different constituent chains differentially perturbed neural crest cell attachment and migration. Sites differentially involved in neural crest cell attachment and migration seemed to be present at the carboxyl termini of the alpha 1(VI) and alpha 2(VI) chains and at the amino-terminus of the alpha 3(VI) chain. The results suggest that neural crest cells interact with Col VI through multiple and cooperative binding sites present within its triple-helical and globular domains. The differential involvement and efficiency of these sites in stimulating neural crest cell adhesion and migration is strongly determined by the supramolecular organization of the collagen and requires inter- and intramolecular structural integrity. Since neural crest cell attachment and migration on Col VI was completely inhibited by anti-beta 1 integrin antibodies, there is evidence that this class of integrins is essential for the neural crest cell--Col VI interaction.

Animals↗

PubMed1994

Characterization of native laminin from bovine kidney and comparison with other laminin variants.

A comprehensive characterization of laminin isoforms requires access to native preparations of laminins of a defined subunit composition. For this purpose an optimized isolation procedure was developed and shown to be broadly applicable to normal mammalian tissues. The protocol does in addition yield side fractions highly enriched in collagens XII and XIV. The major laminin purified from bovine kidney is indistinguishable from mouse Engelbreth-Holm-Swarm (EHS) tumor laminin in electron microscopy, but contains an A chain that migrates in a position intermediate to the Ae and the Am chains on SDS/PAGE. Antisera raised against mouse EHS-tumor laminin crossreact with B chains, but not with the A chain, of kidney laminin. Further, this A chain is not recognized by antisera raised against the Am chain. Laminins from heart and kidney both contain a significant subpopulation with a 190-kDa polypeptide identified as the B1s chain. The Am-containing laminins from heart and placenta differ morphologically from the Ae-containing EHS laminin in having one short arm that does not have the characteristic globule-rod-globule appearance. Further, the Am-containing laminins show a significantly higher thermal stability of the coiled-coil alpha-helical region in the long arm than does Ae-containing EHS laminin, indicating that certain combinations of laminin chains interact more strongly than others.

Animals↗

PubMed1993

Activating mutations of the Gs alpha-gene in nonfunctioning pituitary tumors.

The majority of pituitary tumors are of monoclonal origin; however, the molecular basis for their formation is poorly understood. Somatic mutations in the alpha-subunit of the GTP-binding protein, Gs alpha (gsp oncogene) have been found in about one third of GH-secreting tumors. Mutations in another alpha-subunit of a GTP-binding protein, Gi2 alpha (gip mutations) have been described in other endocrine tumors. In this study, we examined 21 nonfunctioning pituitary tumors and 4 macroprolactinomas for gsp mutations and 27 nonfunctioning tumors and 4 macroprolactinomas for gip mutations. Using the polymerase chain reaction and denaturing gradient gel electrophoresis, 2 nonfunctioning pituitary tumors displayed migration abnormalities when the Gs alpha-gene was analyzed. Sequence analysis of these abnormally migrating polymerase chain reaction products revealed two previously known gsp mutations: arginine at codon 201 altered to cysteine, and glutamine at codon 227 changed to leucine. No gip mutations could be demonstrated. These findings emphasize the monoclonal origin of nonfunctioning pituitary tumors and suggest that cAMP may play a role in tumorigenesis of nonfunctioning pituitary tumors.

Adrenocorticotropic Hormone↗

PubMed1981

IgD-secreting murine plasmacytomas: identification and partial characterization of two IgD myeloma proteins.

We have used an immunofluorescence inhibition assay to identify 2 BALB/c plasmacytomas, TEPC-1017 and TEPC-1033, that secrete large quantitites of IgD. Both TEPC-1017 and TEPC-1033 myeloma proteins bound to anti-kappa as well as hybridoma and heterologous anti-delta antibodies, but not to anti-mu, gamma, alpha, or lambda antibodies. Both myeloma proteins were purified by (NH4)2SO4 precipitation, ion exchange chromatography, gel filtration, and Staphylococcus aureus Protein A absorption. These IgD kappa myeloma proteins were used to prepare affinity purified rabbit antibodies to delta-chain and the TEPC-1017 and TEPC-1033 idiotypes. Native TEPC-1017 and TEPC-1033 both had mobilities between those of mouse IgA kappa dimers and trimers when analyzed by polyacrylamide gradient gel electrophoresis. Both IgD myeloma proteins broke down under mild reducing conditions into subunits with electrophoretic mobilities slightly slower than those of an IgA kappa monomer. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of reduced TEPC-1017 and TEPC-1033 demonstrated kappa-chains and heavy chains that co-migrated with alpha chain. These data suggested that secreted IgD contains 2 delta 2 kappa 2 subunits that are linked by an easily reducible disulfide bond. The kappa-chains of IgD secreted by TEPC-1017 and TEPC-1033 have apparent m.w. of approximately 63,000 daltons, whereas the apparent m.w. of intracytoplasmic delta-chain, intracytoplasmic delta-chain synthesized in the presence of tunicamycin, and the cellfree translation product of TEPC-1017 delta-chain mRNA are 54,000, 43,000, and 44,000 daltons, respectively. This is compatible with the interpretation that the delta-chain peptide has a leader sequence and is N-glycosylated during or shortly after peptide synthesis and is glycosylated further shortly before IgD secretion.

Animals↗

PubMed2004

GABA release and uptake regulate neuronal precursor migration in the postnatal subventricular zone.

In the postnatal subventricular zone (SVZ), astrocyte-like cells tightly encapsulate chains of migrating neuronal precursors, although an influence of the astrocyte-like cells on precursor migration has not yet been demonstrated. Cell migration was studied in acute sagittal brain slices to determine whether GABA signaling between astrocyte-like cells and neuronal precursors controls the speed of neuronal precursor migration in the anterior SVZ and rostral migratory stream of juvenile and adult mice. Application of GABA at 10 microm, a nondesensitizing concentration for GABA(A) receptors (GABA(A)Rs), reduced the rate (mean of approximately 50 microm/hr) of cell migration by 21% via GABA(A)R activation. Application of the GABA(A)R antagonist bicuculline enhanced the migration rate by 30%, suggesting that endogenous GABA tonically reduces the speed of cell migration via GABA(A)R activation. Using immunohistochemistry, we found that astrocyte-like cells express the high-affinity GABA transporter subtype GAT4 on processes ensheathing neuronal precursors that contain GABA. Inhibition of GABA uptake into astrocyte-like cells or enhancement of GABA release from neuronal precursors during high K(+) application further reduced the migration rate by increasing ambient GABA levels. GABA altered the migration speed by interfering with intracellular Ca(2+) signaling independently of cell depolarization, because high K(+) application did not alter the speed of cell migration in the presence of bicuculline. These data indicate that astrocyte-like cells create a microenvironment in which their uniquely positioned GABA transporters control the degree of GABA(A)R activation and the migration of neuronal precursors.

Animals↗

PubMed1984

Phosphorylation in vivo of the P light chain of myosin in rabbit fast and slow skeletal muscles.

The P light chain of myosin is partially phosphorylated in resting slow and fast twitch skeletal muscles of the rabbit in vivo. The extent of P light-chain phosphorylation increases in both muscles on stimulation. Rabbit slow-twitch muscles contain two forms of the P light chain that migrate with the same electrophoretic mobilities as the two forms of P light chain in rabbit ventricular muscle. The rate of phosphorylation of the P light chain in slow-twitch muscle is slower than its rate of phosphorylation in fast-twitch muscles during tetanus. The rate of P light-chain dephosphorylation is slow after tetanic contraction of fast-twitch muscles in vivo. The time course of dephosphorylation does not correlate with the decline of post-tetanic potentiation of peak twitch tension in rabbit fast-twitch muscles. The frequency of stimulation is an important factor in determining the extent of P light-chain phosphorylation in fast- and slow-twitch muscles.

Animals↗

PubMed1991

The RGD containing site of the mouse laminin A chain is active for cell attachment, spreading, migration and neurite outgrowth.

The laminin A chain has been sequenced by cDNA cloning and was found to contain an RGD sequence. Synthetic peptides containing the RGD sequence and flanking amino acids were active in mediating cell adhesion, spreading, migration, and neurite outgrowth. Furthermore, endothelial cell attachment to a laminin substrate was inhibited by an RGD-containing synthetic peptide. Antisera against the integrin (fibronectin) receptor, and monoclonal antibody to the integrin, VLA-6, inhibited cell interaction with laminin, as well as with peptides containing an RGD sequence. These results suggest that the RGD containing site of laminin is active and interacts with the integrin family of receptors in certain cells.

Amino Acid Sequence↗

PubMed1991

Cleavage of a smooth muscle myosin heavy chain near its C terminus by alpha-chymotrypsin. Effect on the properties of myosin.

Limited proteolysis of gizzard myosin by alpha-chymotrypsin converted the heavy chain doublet pattern, seen by gel electrophoresis, to a single band. Light chain degradation was not observed and only minor cleavage occurred at other heavy chain sites. Using a polyclonal antibody raised against a unique sequence from the slower-migrating heavy chain (SM1) it was shown that this conversion was due to the loss of a peptide approximately 4000 daltons from the C terminus of SM1. The peptide was isolated and sequenced, and the cleavage site was identified between phenylalanine 1943 and alanine 1944. Addition of antibody before protease protected SM1 from cleavage. The following changes were observed (a) the Mg2(+)-dependence of actin-activated ATPase of digested phosphorylated myosin was altered and activity was relatively high at low Mg2+ levels, i.e. similar to phosphorylated heavy meromyosin; (b) the KCl dependence of Mg2(+)-ATPase of the digested myosin, particularly the phosphorylated form, showed an altered pattern consistent with the stabilization of the 6 S conformation; (c) the tendency for aggregation was increased by proteolysis of phosphorylated myosin. These results show that the C-terminal region of a gizzard myosin heavy chain can modify some of the properties of myosin. It is suggested that the observed modifications reflect an enhanced tendency of the digested myosin to aggregate.

Actin Cytoskeleton↗

PubMed1978

Collagen synthesis by human amniotic fluid cells in culture: characterization of a procollagen with three identical proalpha1(I) chains.

Second trimester human amniotic fluid cells synthesize and secrete a variety of collagenous proteins in culture. F cells (amniotic fluid fibroblasts) are the most active biosynthetically and synthesize predominantly type I with smaller amounts of type III procollagen. Epithelioid AF cells (the predominating clonable cell type) synthesize a type IV-like procollagen and a procollagen with three identical proalpha chains, structurally and immunologically related to the proalpha1 chains of type I procollagen. The latter procollagen, when cleaved with pepsin and denatured, yields a single non-disulfide-bonded alpha chain that migrates more slowly than F cell or human skin alpha1(I) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis but coelutes with these chains from carboxymethyl-cellulose. The major cyanogen bromide produced peptides demonstrate a similar behavior relative to peptides derived from alpha1(I). The collagen is characterized by an increased solubility at neutral pH and high ionic strength, relative to type I collagen. The amino acid composition of the pepsin-resistant alpha chain is essentially identical with that of human alpha1(I), except for marked increases in the content of 3- and 4-hydroxyproline and hydroxylysine. Preliminary experiments suggest that these increased posttranslational modifications are responsible for the unusually slow migration of this collagen and its cyanogen bromide peptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The procollagen has, therefore, been assigned the chain composition [proalpha1(I)]3. Like type I procollagen, [proalpha1(I)]3 undergoes a time-dependent conversion, in the medium and cell layer, to procollagen intermediates and alpha chains. The production of [proalpha1(I)]3 probably reflects the state of differentiation and/or embryologic derivation of AF cells rather than a characteristic of the fetal phenotype, since F cells do not synthesize significant amounts of the procollagen.

Amino Acids↗

PubMed1989

A synthetic peptide containing the IKVAV sequence from the A chain of laminin mediates cell attachment, migration, and neurite outgrowth.

Laminin is a basement membrane glycoprotein which consists of A, B1, and B2 chains. Laminin has diverse biological activities including promoting cell adhesion, migration, differentiation, growth, and neurite extension. Synthetic peptides from the active region of the A chain were prepared and tested for their biological activity. A 19-mer peptide (designated PA22-2), from just above the carboxyl globule on the long arm of the A chain, was found to promote cell adhesion, spreading, migration, and neurite outgrowth. By testing smaller sequences within the 19-mer peptide, a constituent pentapeptide, IKVAV (Ile-Lys-Val-Ala-Val), was identified as the active site for cell adhesion and neurite outgrowth. These data suggest that this sequence is one of the principle sites in laminin which regulate cellular behavior.

Adrenal Gland Neoplasms↗

PubMed1997

Mechanism of migration of olfactory bulb interneurons.

Neuroblasts in the subventricular zone of the walls of the lateral ventricle in the brain of young and adult rodents migrate into the olfactory bulb where they differentiate into local interneurons. These cells move closely associated with each other, forming chains without radial glial or axonal guidance. The migrating neuroblasts express PSA-NCAM on their surface and PSA residues are crucial for cell-cell interaction during chain migration. This migration occurs throughout the lateral wall of the lateral ventricle, where the precursors form an extensive network of chains. Cells remain organized as chains until they reach the olfactory bulb, where they disperse organized as chains until they reach the olfactory bulb, where they disperse radially as individual cells. Chain migration defines a novel form of neuronal precursor translocation which is based on homotypic interactions between cells.

Journal Article↗

PubMed1975

Synthesis of myosin heavy and light chains in muscle cultures.

The weight ratio of myosin/actin, the myosin heavy chain content as the percentage of total protein (wt/wt), and the kinds of myosin light chains were determined in (a) standard muscle cultures, (b) pure myotube cultures, and (c) fibroblast cultures. Cells for these cultures were obtained from the breast of 11-day chick embryos. Standard cultures contain, in addition to myotubes, large numbers of replicating mononucleated cells. By killing these replicating cells with cytosine arabinoside, pure myotube cultures were obtained. The myosin/actin ratio (wt/wt) for pure myotube, standard muscle, and fibroblast cultures average 3.1, 1.9, and 1.1 respectively. By day 7, myosin in myotube cultures represents a minimum of 7% of the total protein, but about 3% in standard cultures and less than 1.5% in fibroblasts cultures. Myosin from standard cultures contains light chain LC1, LC2, and LC3, with a relative stoichiometry of the molarity of 1.0:1.9:0.5 and mol wt of 25,000, 18,000 and 16,000 daltons, identical to those in adult fast muscle. Myosin from pure myotubes exhibits light chains LC1 and LC2, with a molar ratio of 1.5:1.6. Myosin from fibroblast cultures possesses two light chains with a stoichiometry of 1.8:1.8 and mol wt of 20,000 and 16,000 daltons. Clearly, the faster migrating light chain, LC3, found in standard cultures is synthesized not by the myotubes but ty the mononucleated cells. In myotubes, both the assembly of the sarcomeres and the interaction between thick and thin filaments required for spontaneous contraction occur in the absence of light chain LC3. One set of structural genes for the myosin light and heavy chains appears to be active in mononucleated cells, whereas another set appears to be active in multinucleated myotubes.

Adenosine Triphosphatases↗

PubMed2003

Characterization and quantitation of mixtures of alkyl ether sulfates and carboxylic acids by capillary electrophoresis with indirect photometric detection.

The separation, characterization, and determination of mixtures of alkyl ether sulfates (AES) and fatty acids (C10-C16) in background electrolytes (BGEs) containing acetonitrile (ACN)-water mixtures is addressed. Due to inhibition of the ionization of the carboxylate groups, the migration time and the resolution between the fatty acids decreased when the water content of the BGE was reduced, but efficiency and resolution between the AES oligomers improved. The migration times increased and resolution improved by substituting 5% ACN by an equivalent amount of dioxane. A complete separation of the two surfactant classes, up to the AES oligomers with 8 ethylene oxide units (EOs) with respect to C10, with excellent resolution between the AES oligomers, while preserving a satisfactory resolution between the fatty acids, was achieved with a BGE containing 5 mM trimethoxybenzoic acid, 7 mM dipentylamine, 85% ACN, 5% dioxane, and 10% water. The two surfactant classes were increasingly resolved by further reducing the water content of the BGE. Thus, C2 (acetate) was resolved from the AES oligomers up to 7 EOs using 90% ACN and 5% dioxane, but the resolution between the heavier fatty acids was poor with this BGE. Identification of the AES oligomers was eased by the excellent regularity of the successive migration times; thus, within each AES subclass or series of oligomers with the same number of carbon atoms in the alkyl chain, the migration times decreased following a mild curve as the number of EOs increased. The way how the data obtained by indirect photometry (corrected peak areas that are proportional to the molar concentrations) should be managed to avoid systematic error when the calibration curve is constructed using an AES standard with an oligomer distribution different from that of the samples is discussed and equations are given. Decyl sulfate was successfully used as internal standard. The detection limits (S/N = 3) were of ca. 2 microM for individual AES oligomers.

Alkanesulfonates↗

PubMed2002

Role of D and E domains in the migration of vascular smooth muscle cells into fibrin gels.

The structure of fibrin plays an important role in the organization of thrombi, the development of atherosclerosis, and restenosis after PTCA. In this study, we examined the mechanisms of the migration of vascular smooth muscle cells (SMCs) into fibrin gels, using an in vitro assay system. Cultured SMCs from bovine fetal aortic media migrated into fibrin gels prepared with thrombin, which cleaves both fibrinopeptides A and B from fibrinogen, without other chemotactic stimuli. Both desA fibrin gels prepared with batroxobin, which cleaves only fibrinopeptide A, and desB fibrin gels prepared with Agkistrodon contortrix thrombin-like enzyme (ACTE), which cleaves only fibrinopeptide B, similarly induced the migration of SMCs compared to fibrin gels prepared with thrombin. These results suggest that the cleavage of fibrinopeptides is not necessary, but rather that the three-dimensional structure of the gel may be important for the migration of SMCs. Furthermore, gels prepared with protamine sulfate, which forms fibrin-like gels non-enzymatically, similarly induced the migration of SMCs compared to the gels prepared with thrombin. Both anti-fibrin(ogen) fragment D and anti-fibrin(ogen) E antibodies inhibited the migration of SMCs into fibrin gels, suggesting that both the D and E domains of fibrin(ogen) are involved in the migration of SMCs into fibrin gels. The addition of GRGDS, a synthetic RGD-containing peptide, but not that of GRGES, a control peptide, partially inhibited the migration of SMCs into fibrin gels, suggesting that the migration of SMCs into fibrin gels is at least in part dependent on the RGD-containing region of the alpha chain. The migration of SMCs into fibrin gels was also inhibited by a monoclonal antibody for integrin alpha v beta 3 and alpha 5 beta 1, indicating that migration is dependent on these integrins. Furthermore, both fibrin(ogen) fragments D and E inhibited the migration of SMCs into fibrin gels, suggesting that these fragments, generated during fibrino(geno)lysis, may be relevant in the regulation of SMC migration into fibrin gels.

Animals↗

PubMed2003

Coexpression of matrilysin and laminin-5 gamma2 chain may contribute to tumor cell migration in colorectal carcinomas.

We attempted to examine the correlation between matrilysin and laminin-5 gamma2 chain expression with reference to the number of dedifferentiation units along the entire invasive front (tumor budding). Immunostaining for hMMP-7 and laminin-5 gamma2 chain was performed in 50 T1 colorectal carcinomas, and immunoreactivity was evaluated at the invasive front of the tumor. On hematoxylin-eosin sections, the number of tumor budding was counted. The localization of matrilysin tended to be widespread compared with that of laminin-5 gamma2 chain. Matrilysin and laminin-5 gamma2 chain expression were positive in 28 (56%) and 15 (30%) tumors respectively. There was a significant correlation between matrilysin and laminin-5 gamma2 chain expression (P = 0.02). Matrilysin(+)/laminin-5 gamma2 chain(+) tumors had a significantly greater amount of tumor budding than matrilysin(-)/laminin-5 gamma2 chain(-) tumors (P = 0.003) or matrilysin(+)/laminin-5 gamma2 chain(-) tumors (P = 0.03). In conclusions, coexpression of matrilysin and laminin-5 gamma2 chain may contribute to tumor cell migration in colorectal carcinomas.

Adult↗

PubMed2006

Role of myosin regulatory light chain and Rac1 in the migration of polyamine-depleted intestinal epithelial cells.

We have previously shown that polyamine depletion decreased migration, Rac activation, and protein serine threonine phosphatase 2A activity. We have also shown that polyamine depletion increased cortical F-actin and decreased lamellipodia and stress fibers. In this study, we used staurosporine (STS), a potent, cell-permeable, and broad-spectrum serine/threonine kinase inhibitor, and studied migration. STS concentrations above 100 nM induced apoptosis. However, in polyamine-depleted cells, a lower concentration of STS (5 nM) increased attachment, spreading, Rac1 activation, and, subsequently, migration without causing apoptosis. STS-induced migration was completely prevented by a Rac1 inhibitor (NSC-23766) and dominant negative Rac1. These results imply that STS restores migration in polyamine-depleted cells through Rac1. The most important finding in this study was that polyamine depletion increased the association of phosphorylated myosin regulatory light chain (pThr(18)/Ser(19)-MRLC) at the cell periphery, which colocalized with thick cortical F-actin. Localization of pThr(18)- and pSer(19)-MRLC was found with stress fibers and nuclei, respectively. STS decreased the phosphorylation of cellular and peripheral pThr(18)-MRLC without any effect on nuclear pSer(19)-MRLC, dissolved thick cortical F-actin, and increased lamellipodia and stress fiber formation in polyamine-depleted cells. In control and polyamine-depleted cells, focal adhesion kinase (FAK) colocalized with stress fibers and the actin cortex, respectively. STS reorganized FAK, paxillin, and the cytoskeleton. These results suggest that polyamine depletion prevents the dephosphorylation of MRLC and thereby prevents the dynamic reorganization of the actin cytoskeleton and decreases lamellipodia formation resulting in the inhibition of migration.

Actins↗

PubMed1987

Limited proteolysis of type I collagen at hyperreactive sites by class I and II Clostridium histolyticum collagenases: complementary digestion patterns.

The initial proteolytic events in the hydrolysis of rat tendon type I collagen by the class I and II collagenases from Clostridium histolyticum have been investigated at 15 degrees C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been used to detect the initial cleavage fragments of both the alpha 1(I) and alpha 2 chains, which migrate at different rates in the buffer system employed. Experiments with the class I collagenases indicate that the first cleavage occurs across all three chains of the triple helix close to the C-terminus to produce fragments whose alpha chains have molecular weights of approximately 88,000. The second cleavage occurs near the N-terminus to reduce the molecular weight of the alpha chains to 80,000. Initial proteolysis by the class II collagenases occurs across all three chains at a site in the interior of the collagen triple helix to give N- and C-terminal fragments with alpha-chain molecular weights of 35,000 and 62,000, respectively. The C-terminal fragment is subsequently cleaved to give fragments with alpha-chain molecular weights of 59,000. These results indicate that type I collagen is degraded at several hyperreactive sites by these enzymes. Thus, initial proteolysis by these bacterial collagenases occurs at specific sites, much like the mammalian collagenases. These results with the individual clostridial collagenases provide an explanation for earlier data which indicated that collagen is degraded sequentially from the ends by a crude clostridial collagenase preparation.

Clostridium↗