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PubMedDate not supplied

Histochemical studies of epithelial cell glycoproteins in normal rat colon.

Two general classes of glycoproteins have been identified in the colonic epithelial cells of the Sprague Dawley rat. Glycoproteins belonging to the first of these classes contain sialic acids both with and without side chain o-acyl substituents, abundant o-sulphate ester and 'neutral sugars' (hexose, 6-deoxyhexose or N-acetyl hexosamine residues) with oxidisable vicinal diols and are located in the goblet cells of the descending colon and in goblet cells populating the upper halves of the crypts of the ascending colon. In the descending colon, the sulphosialoglycoproteins in the goblet cells in the base of the crypts contain sialic acids without side chain o-acyl substituents. It appears that as these cells migrate up the crypts, there is o-acylation of the side chains of the sialic acids of the glycoproteins and an increase in the quantity of 'neutral sugars' without a corresponding increase in sialic acid. Glycoproteins with similar properties to those of the goblet cells of the upper halves of the crypts of the descending colon, but containing less sulphate, are found in the goblet cells of the upper half of the crypts of the ascending colon. The second general class of glycoproteins contain sialic acids all, or almost all of which, are substituted at position C8 and only relatively small quantities of sulphate. They are located in the mucous cells of the descending colon, the deep crypt secretory cells of the ascending colon and the columnar absorptive cell brush border.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

Normal and defective neuronal membranes: structure and function: neuronal lesions in peroxisomal disorders.

Neuronal involvement in the peroxisomal disorders is divided into two main groups: developmental and postdevelopmental or degenerative. In the former the major lesions are neuronal migration abnormalities, which vary from severe in the cerebro-hepato-renal (Zellweger) syndrome (ZS) to mild in neonatal adrenoleukodystrophy. More common, but much less severe, are defects in neuronal differentiation or terminal migration, particularly involving the inferior medullary olives. Ultrastructural and neurochemical observations in ZS suggest that the presence of abnormal cytosomes in migrating neurons and radial glia, probably the result of excessive very long chain fatty acids, are responsible in part for its major neocortical migration defect, parasylvian pachygyria-polymicrogyria. The postdevelopmental neuronal lesions involve specialized sensory neurons of the retina and the inner ear, resulting in atypical retinitis pigmentosa and its consequent visual defects and sensorineural hearing deficits. Neuronal atrophy and/or loss is seen in both the dorsal-root ganglia of adrenomyeloneuropathy and the atrophic cerebellum of rhizomelic chondodysplasia punctata. The underlying pathophysiology of these neuronal lesions is postulated to be caused by the incorporation of abnormal fatty acids into neuronal membranes, leading to an unresponsiveness to neurotrophic factors necessary for normal function and survival or to increased permeability of calcium channels and cell death.

Adrenoleukodystrophy↗

PubMed2007

The beta3 chain short arm of laminin-332 (laminin-5) induces matrix assembly and cell adhesion activity of laminin-511 (laminin-10).

The basement membrane (BM) protein laminin-332 (Lm332) (laminin-5) has unique activity and structure as compared with other laminins: it strongly promotes cellular adhesion and migration, and its alpha3, beta3, and gamma2 chains are all truncated in their N-terminal regions (short arms). In the present study, we investigated the biological function of the laminin beta3 chain. When the beta3 chain short arm (beta3SA) was overexpressed in HEK293 cells (beta3SA-HEK), they deposited a large amount of beta3SA and a small amount of laminin-511 (Lm511) (laminin-10) on culture plates. Control HEK293 cells secreted Lm511 but failed to deposit it. The extracellular matrix (ECM) deposited by beta3SA-HEK cells strongly promoted cell attachment and spreading. The beta3SA-HEK ECM did not directly bind Lm511, but it stimulated control HEK293 cells to deposit Lm511 on the culture plates. Although purified beta3SA did not support cell adhesion by itself, it enhanced the cell adhesion activity of Lm511. Experiments with anti-integrin antibodies also suggested that the strong cell adhesion activity of the beta3SA-HEK ECM was derived from the synergistic action of beta3SA and Lm511. It has previously been found that beta3SA binds an unknown cell surface receptor. Taken together, the present study suggests that the short arm of the laminin beta3 chain enhances the matrix assembly of Lm511 and its cell adhesion activity by interacting with its receptor.

Blotting, Western↗

PubMed1997

Maturation and migration of murine dendritic cells in situ. Observations in a skin organ culture model.

Dendritic cells reside in tissues such as skin in an immature state. Upon antigenic challenge they begin to mature and migrate to the draining lymph nodes. These processes are still poorly understood. One way to study in situ aspects of maturation and migration are skin organ culture models. In an attempt to learn more about the relationship between maturation and migration we investigated the expression of several marker molecules by immunohistochemistry. Sheets from normal murine ear skin and from skin that had been cultured for three days were compared. During culture the numbers of epidermal Langerhans cells decreased and accumulations of strongly MHC class II-positive cells ("cords") were found in the dermis. As compared to untreated skin, the few Langerhans cells remaining in the epidermal sheets after 2-3 days expressed increased levels of MHC class II and had also upregulated B7-2 (CD86) as described. They did not express the antigen recognized by mAb 2A1, a marker for mature dendritic cells. Double-staining of dermal sheets after 3 days of culture showed that dendritic cells in the "cords" expressed high levels of MHC class II and CD86 but were also reactive with mAb 2A1. This pattern is identical to those dendritic cells that had emigrated into the culture medium over the period of 3 days. Invariant chain (mAb In1) was detected at all stages of culture as opposed to isolated epidermal Langerhans cells in suspension where invariant chain expression disappears after 3 days of culture. We conclude that the up- (class II, B7-2, 2A1) and down-regulation (invariant chain) of dendritic cell molecules during migration does not happen in a synchronized manner. The molecule recognized by mAb 2A1 seems to appear late in maturation.

Animals↗

PubMed1995

Factors XWenatchee I and II: compound heterozygosity involving two variant proteins.

Variant factor X in an individual with a mild bleeding tendency was suspected based on deficient procoagulant activity (10-20% of normal) and antigen (30-35% of normal) levels of plasma factor X. Heteroduplex analysis of factor X gene exons indicated heterozygosity for mutations in both exons 6 and 4, confirmed by direct sequencing of the amplified exons. Substitution of C by T at nucleotide position 13,984 (Arg-139 to Cys) was found in the factor X gene exon 6 of the propositus. This mutation creates a BsmI site and the patient tested heterozygous for the BsmI cleavage involved, as did one of his two daughters. In addition, exon 4 was found to have the normal A and a novel C (Asn-57 to Thr) at nucleotide position 9338. The exon 4 mutation creates a BsaJI site, detectable after amplification mismatch to remove an existing BsaJI site. Both the patient and the second of his two daughters were heterozygous for this cleavage. The two variant proteins are called factors XWenatchee I (Arg-139 to Cys) and II (Asn-57 to Thr). A mixed variant isolate derived from the plasma of the propositus exhibited heavy/light chains of normal size, as well as an apparent single-chain molecule not dissociable by reducing agent. A single-chain molecule would be predicted for form I, if the mutation blocks processing cleavages that normally remove a tripeptide interposed between the heavy and light chains. A Western blot of partially purified factor X from the daughter who inherited the form I defect revealed a component migrating the same as the putative single-chain species. Based upon the factor X activity vs. antigen ratios for the propositus and both daughters, both forms I and II are probably dysfunctional molecules.

Amino Acid Sequence↗

PubMed1980

An improved procedure for the preparation of alkyl sulphate esters suitable for the study of secondary alkylsulphohydrolase enzymes.

A simple, rapid and convenient method for the synthesis of secondary alkyl sulphate esters is described in which the sodium alkoxide of the parent alcohol is sulphated by using triethylamine-SO3 complex. The procedure gives relatively good yields, even for the sulphation of long-chain alcohols and those in which the hydroxy group is remote from the terminal carbon atoms. Positional isomerization, arising from the migration of the hydroxy group along the carbon chain, is absent, and resolved enantiomers of alcohols react with complete retention of configuration.

Alcohols↗

PubMed2004

Structural and functional basis of the serine protease-like hepatocyte growth factor beta-chain in Met binding and signaling.

Hepatocyte growth factor (HGF), a plasminogen-related growth factor, is the ligand for Met, a receptor tyrosine kinase implicated in development, tissue regeneration, and invasive tumor growth. HGF acquires signaling activity only upon proteolytic cleavage of single-chain HGF into its alpha/beta heterodimer, similar to zymogen activation of structurally related serine proteases. Although both chains are required for activation, only the alpha-chain binds Met with high affinity. Recently, we reported that the protease-like HGF beta-chain binds to Met with low affinity (Stamos, J., Lazarus, R. A., Yao, X., Kirchhofer, D., and Wiesmann, C. (2004) EMBO J. 23, 2325-2335). Here we demonstrate that the zymogen-like form of HGF beta also binds Met, albeit with 14-fold lower affinity than the protease-like form, suggesting optimal interactions result from conformational changes upon cleavage of the single-chain form. Extensive mutagenesis of the HGF beta region corresponding to the active site and activation domain of serine proteases showed that 17 of the 38 purified two-chain HGF mutants resulted in impaired cell migration or Met phosphorylation but no loss in Met binding. However, reduced biological activities were well correlated with reduced Met binding of corresponding mutants of HGF beta itself in assays eliminating dominant alpha-chain binding contributions. Moreover, the crystal structure of HGF beta determined at 2.53 A resolution provides a structural context for the mutagenesis data. The functional Met binding site is centered on the "active site region" including "triad" residues Gln(534) [c57], Asp(578) [c102], and Tyr(673) [c195] and neighboring "activation domain" residues Val(692), Pro(693), Gly(694), Arg(695), and Gly(696) [c214-c219]. Together they define a region that bears remarkable resemblance to substrate processing regions of serine proteases. Models of HGF-dependent Met receptor activation are discussed.

Amino Acid Sequence↗

PubMed1998

Neural crest cell dynamics revealed by time-lapse video microscopy of whole embryo chick explant cultures.

DiI-labeled cranial neural crest cells were followed in whole embryo chick explant cultures using time-lapse confocal microscopy. Neural crest cells emerged along the dorsal midline of all rhombomeres. There was a small amount of mixing of neural crest cells between adjoining rhombomeres as cells emerged from the dorsal midline; this mixing persisted during their migration out of the neural tube. Neural crest cell-free zones lateral to rhombomere 3 (r3) and r5 resulted from neural crest cells migrating in either rostral or caudal directions to join other neural crest cells exiting adjacent to r2, r4, or r6. Neural crest cells migrated in a wide variety of individual cell behaviors, ranging from rapid unidirectional motion to stationary and even backward movement (toward the neural tube). Neural crest cells also migrated collectively, extending filipodia to form chain-like cell arrangements. In the midbrain and r1 region, many chains stretched from the dorsal midline to just beyond the lateral extent of the neural tube. In the r7 region, cells linked together and stretched laterally from the neural tube to other neural crest cells migrating into the third branchial arch. The unpredictable cell trajectories, the mixing of neural crest cells between adjoining rhombomeres, and the diversity in cell migration behavior within any particular region imply that no single mechanism guides migration. The regional differences in cell migration characteristics suggests that influential factors may vary spatially along the rostrocaudal axis in the head.

Animals↗

PubMed1990

Inhibition of neural crest cell migration by aggregating chondroitin sulfate proteoglycans is mediated by their hyaluronan-binding region.

We have recently shown that the large hyaluronan-aggregating chondroitin sulfate proteoglycan from cartilage (PG-LA) is unfavorable as a substrate for neural crest cell migration in vitro and that this macromolecule inhibits cell dispersion on fibronectin substrates when included in the medium (R. Perris and S. Johansson, 1987, J. Cell Biol. 105, 2511-2521). In this study we present data on the specificity of the migration-repressing activity of PG-LA and data on the molecular mechanisms by which the proteoglycan might impair neural crest cell motility. Soluble PG-LA potently impaired cell migration on substrates of laminin/laminin-nidogen, vitronectin, and collagen types I, III, IV, and VI. When tested in solid-phase binding assays, PG-LA bound avidly to substrates of collagen types I-III and V. Conversely, minimal amounts of the proteoglycan bound to substrates of laminin-nidogen, vitronectin, collagen types IV and VI, and fibronectin or to a proteolytic fragment encompassing its cell-binding domain (105 kDa). Preincubation of these substrates with soluble PG-LA prior to plating of the cells had no effect on their locomotory behavior. These results indicate that PG-LA affects neural crest cell movement primarily through an interaction with the cell surface, rather than by association with the cell motility-promoting substrate molecules. The molecular interaction of soluble PG-LA with neural crest cells was further examined by analyzing the effects of isolated domains of the proteoglycan on cell migration on fibronectin. Addition of chondroitin sulfate chains, the core protein free of glycosaminoglycans, the isolated hyaluronan-binding region (HABr), or a proteolytic fragment corresponding to the keratan sulfate-enriched domain of the PG-LA to neural crest cells migrating on fibronectin or the 105-kDa fibronectin fragment had no significant effect on their motility. After reduction and alkylation, PG-LA was considerably less efficient in perturbing cell movement on fibronectin substrates and virtually ineffective in altering migration on the 105-kDa fragment. In the presence of hyaluronan fragments of 16-30 monosaccharides in length, or an antiserum against the HABr, the migration repressing activity of PG-LA was reduced in a dose-dependent fashion. Furthermore, the inhibitory action of PG-LA was significantly reduced by treatment of the cells with Streptomyces hyaluronidase.(ABSTRACT TRUNCATED AT 400 WORDS)

Ambystoma↗

PubMed2005

IgE- and FcepsilonRI-mediated migration of human basophils.

Local accumulation of basophils at inflammatory sites is observed in experimental antigen challenge and in allergic diseases. It is not fully known what factor(s) regulates local basophil influx in tissues, and it has not been determined whether antigens belong in a panel of basophil chemoattractants. This study was designed to elucidate whether IgE- and high-affinity receptor for IgE (FcepsilonRI)-mediated stimulation can induce human basophil migration. The migration-inducing potency of an anti-FcepsilonRI alpha-chain mAb, CRA-1, was examined on human basophils. CRA-1 mAb elicited significant migration of basophils. The migration-inducing potency of this mAb was maximal at 100 ng ml-1, and CRA-1 mAb at 100 ng ml-1 attracted approximately 10% of total inoculated basophils above baseline levels after incubation for 2.5 h. Checkerboard analysis indicated that basophil migration induced by this mAb was mainly chemotactic and partially chemokinetic. An antigen, Der f 2, also induced migration of basophils from Der f-sensitive subjects. Basophils mixed with 1 ng ml-1 of CRA-1 mAb showed an exaggerated migration response to eotaxin, indicating that FcepsilonRI cross-linkage enhances basophil migration to other chemoattractants. Induction of basophil migration by IgE- and FcepsilonRI-cross-linking stimulation may, at least in part, explain the pathogenesis of local basophil accumulation clinically observed in allergic diseases such as asthma.

Antigens, Dermatophagoides↗

PubMed2005

Heat shock protein 60 activates cytokine-associated negative regulator suppressor of cytokine signaling 3 in T cells: effects on signaling, chemotaxis, and inflammation.

Previously, we reported that treatment of T cells with the 60-kDa heat shock protein (HSP60) inhibits chemotaxis. We now report that treatment of purified human T cells with recombinant human HSP60 or its biologically active peptide p277 up-regulates suppressor of cytokine signaling (SOCS)3 expression via TLR2 and STAT3 activation. SOCS3, in turn, inhibits the downstream effects of stromal cell-derived-1alpha (CXCL12)-CXCR4 interaction in: 1) phosphorylation of ERK1/2, Pyk2, AKT, and myosin L chain, required for cell adhesion and migration; 2) formation of rear-front T cell polarity; and 3) migration into the bone marrow of NOD/SCID mice. HSP60 also activates SOCS3 in mouse lymphocytes and inhibits their chemotaxis toward stromal cell-derived factor-1alpha and their ability to adoptively transfer delayed-type hypersensitivity. These effects of HSP60 could not be attributed to LPS or LPS-associated lipoprotein contamination. Thus, HSP60 can regulate T cell-mediated inflammation via specific signal transduction and SOCS3 activation.

Adoptive Transfer↗

PubMed1992

Lymphocyte activation and regulation of three adhesion molecules with supposed function in homing: LECAM-1 (MEL-14 antigen), LPAM-1/2 (alpha 4-integrin) and CD44 (Pgp-1).

Directed migration of lymphocytes from blood into lymph nodes and gut-associated lymphatic tissue, also referred to as homing, is subject to change following activation. Lymphocyte migration into lymphoid organs in vivo and binding to high endothelial venules in vitro is largely suppressed after short-term stimulation with phorbol esters. The observed functional alterations were correlated with changes in the expression of three putative homing receptors, LECAM-1 (MEL-14 antigen), LPAM-1/2 (alpha 4-integrin) and the murine CD44 (Pgp-1, H-CAM, Hermes-antigen equivalent) upon different modes of cellular activation. Expression of LECAM-1 (gp90 MEL-14), a lymphocyte adhesion molecule implicated in targeting extravasation into lymph nodes, was found to be lost almost completely within minutes after protein kinase C activation. LECAM-1 re-expression occurred within less than 24 h. Rapid loss of LECAM-1 was also observed after calcium ionophores whereas anti-CD3 or concanavalin A elicited a gradual and heterogeneous loss of LECAM-1 becoming detectable after several hours only. A number of cytokines tested were not able to induce alterations in LECAM-1 expression. In contrast, expression of LPAM-1/2 (alpha 4-integrin) and CD44 (Pgp-1, H-CAM), two adhesion molecules supposed to direct extravasation into Peyer's patches, remained stable for hours after every stimulus tested; CD44 expression gradually increased 24 h after mitogenic activation, whereas a small reduction only was observed for the expression of the alpha 4-chain under certain conditions. Thus, reduced extravasation of lymphocytes into Peyer's patches after activation is not due to a decline in the surface density of LPAM-1/2 alpha-chain or CD44 whereas alterations in migration into lymph nodes parallel the expression of LECAM-1.

Animals↗

PubMed1993

Hydride shifts in the biosynthesis of the p-menthane monoterpenes alpha-terpinene, gamma-terpinene, and beta-phellandrene.

The biosynthesis of several monoterpenes from the acyclic precursor geranyl pyrophosphate requires the migration of positive charge from the isopropyl side chain into the cyclohexenoid ring of the universal alpha-terpinyl cation intermediate of the reaction. The hydride shifts responsible for charge migration in the formation of three p-menthane olefin isomers were examined in a range of plant species: alpha-terpinene in American wormseed, gamma-terpinene in thyme, and (-)-beta-phellandrene in lodgepole pine. The experimental approach utilized soluble cell-free enzyme systems and specifically labeled geranyl substrates, with the determination of the labeling patterns in the resulting cyclic products. The results were consistent with stereoelectronic features of the cyclization and support the general model for monoterpene formation.

Cell-Free System↗

PubMed1994

Characterization of type II and type XI collagen synthesis by an immortalized rat chondrocyte cell line (IRC) having a low level of type II collagen mRNA expression.

The biosynthesis of type XI and type II collagens was examined using a stable rat chondrocyte cell line established by W. E. Horton et al. (1988, Exp. Cell Res. 178, 457-468.). These cells (IRC; immortalized rat chondrocytes) were created by transformation with a murine retrovirus carrying the v-myc and v-raf oncogenes. They grow in suspension culture as multicellular aggregates and synthesize typical cartilage proteins, aggrecan and link protein. Type II collagen is absent or synthesized at severely reduced levels, as shown by Northern analysis of mRNA. Thus, this cell type represents a unique model in which to study cartilage matrix protein interactions in the absence of type II collagen. A more detailed look at the proteins secreted into the medium by metabolically labeled IRC cells revealed the presence of collagenase-sensitive bands when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bands were identified as the alpha 1, alpha 2, and alpha 3 chains of heterotrimeric type XI collagen by electrophoretic migration after pepsin digestion, by CNBr peptide mapping, and by immunoprecipitation with antibodies to rat alpha 1(XI). mRNA for all three chains was detected by Northern blot analysis. The data indicate that the low level of alpha 1(II) mRNA previously detected in these cells is translated into pro alpha 3(XI) polypeptide chains which are incorporated into molecules of type XI. Under normal culture conditions, homotrimers of type II collagen were not detected. The carboxyl propeptide domain of the fibrillar collagens directs chain selection and molecular assembly of the trimeric molecules. The sequence of the carboxyl propeptide domain from pro alpha 3(XI) of IRC cells was found to be identical to this domain from pro alpha 1(II) of swarm rat chondrosarcoma, supporting previous evidence that pro alpha 3(XI) and pro alpha 1(II) have the same primary structure. When cultured in the presence of 50 mM arginine, IRC cells could be induced to synthesize pro alpha 1(II) chains in excess over pro alpha 1(XI) and pro alpha 2(XI). Only under these conditions were type II collagen molecules detected, suggesting a preferential association of pro alpha 1(II) with the pro alpha 1 and/or pro alpha 2 chains of type XI collagen.

Amino Acid Sequence↗

PubMed1998

The involvement of the fibronectin type II-like modules of human gelatinase A in cell surface localization and activation.

Recombinant collagen-binding domain (rCBD) comprising the three fibronectin type II-like modules of human gelatinase A was found to compete the zymogen form of this matrix metalloproteinase from the cell surface of normal human fibroblasts in culture. Upon concanavalin A treatment of cells, the induced cellular activation of gelatinase A was markedly elevated in the presence of the rCBD. Therefore, the mechanistic aspects of gelatinase A binding to cells by this domain were further studied using cell attachment assays. Fibroblasts attached to rCBD-coated microplate wells in a manner that was inhibited by soluble rCBD, blocking antibodies to the beta1-integrin subunit but not the alpha2-integrin subunit, and bacterial collagenase treatment. Addition of soluble collagen rescued the attachment of collagenase-treated cells to the rCBD. As a probe on ligand blots of octyl-beta-D-thioglucopyranoside-solubilized cell membrane extracts, the rCBD bound 140- and 160-kDa protein bands. Their identities were likely procollagen chains being both bacterial collagenase-sensitive and also converted upon pepsin digestion to 112- and 126-kDa bands that co-migrated with collagen alpha1(I) and alpha2(I) chains. A rCBD mutant protein (Lys263 --> Ala) with reduced collagen affinity showed less cell attachment, whereas a heparin-binding deficient mutant (Lys357 --> Ala), heparinase treatment, or heparin addition did not alter attachment. Thus, a cell-binding mechanism for gelatinase A is revealed that does not involve the hemopexin COOH domain. Instead, an attachment complex comprising gelatinase A-native type I collagen-beta1-integrin forms as a result of interactions involving the collagen-binding domain of the enzyme. Moreover, this distinct pool of cell collagen-bound proenzyme appears recalcitrant to cellular activation.

Antibodies↗

PubMed1988

Selection of variable-joining region combinations in the alpha chain of the T cell receptor.

Most T lymphocytes express an antigen-specific receptor composed of two subunits, alpha and beta, each of which can exhibit structural variability. A complex selection process operates on T cells during development in the thymus such that cells expressing only particular alpha beta-receptors migrate to the periphery. The alpha-chain repertoire was dissected at different stages of the selection process by using the polymerase chain reaction (PCR) technique to amplify only those transcripts of a particular variable region gene (V58). Sequences from these V58 cDNAs reveal the predominant expression of four joining (J) segments by T cells in the adult thymus, suggesting that molecular or cellular processes select particular V alpha J alpha combinations during development. T cells expressing one of these V58J alpha chains appear to have been negatively selected at a later stage, since these transcripts were present in the spleen at approximately one-tenth the level in the thymus. Results also indicate that residues present at the V alpha J alpha junction may be important in an early selection process.

Animals↗

PubMed1982

Hemidesmosomes of normal and regenerating mouse corneal epithelium.

Hemidesmosomes of normal mouse corneal epithelium observed in tangential thin sections, occupy 14% of the basal plasma membrane. They consist of linear chains of densities with an orientation that is not random with respect to the radial axis of the cornea, tending to parallel it. During the repair of a small epithelial defect, cells of the corneal epithelium peripheral to the defect show chains of hemidesmosomes arranged parallel to the direction of migration of the epithelial sheet. This is parallel to the radius, like the orientation of the normal chains. Cells of the area that was denuded of epithelium, and is being resurfaced, show no hemidesmosomes. During repair of a large defect of the corneal epithelium hemidesmosomes are present on the cells covering the denuded area but they are small, few in number compared to the normal, and many are not arranged in chains. These small hemidesmosomes appear to be points of attachment of very fine basal filaments, possibly actin.

Animals↗

PubMed1996

Differential neural crest cell attachment and migration on avian laminin isoforms.

A number of laminin isoforms have recently been identified and proposed to exert different functions during embryonic development. In the present study, we describe the purification and partial characterization of several isoforms isolated from chick heart and gizzard, and provide data on the molecular mechanisms underlying the interaction of avian neural crest cells with these molecules in vitro. Laminins extracted from heart and gizzard tissues were separated by gel filtration and purified to homogeneity by sequential lectin and immunoaffinity chromatography by utilizing monoclonal antibodies directed against the avian alpha 2, beta 2 and gamma 1 laminin chains. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) banding pattern of the polypeptide complexes obtained and immunoblotting with polyclonal antisera allowed the identification of Laminin-2 (alpha 2 beta 1 gamma 1), Laminin-4 (alpha 2 beta 2 gamma 1), and laminins comprising the beta 1, beta 2 and gamma 1 chains associated with a shorter alpha chain which, in SDS-PAGE, co-migrate with the beta/gamma complex in the 200 kDa region. These latter laminins, which are here arbitrarily denoted Laminin-alpha x (heart tissue) and Laminin-G (gizzard tissue), are somewhat distinct in their apparent molecular weight, are differentially associated with nidogen, and appear as "T"-shaped particles similar to Laminin-6 and Laminin-7 when analyzed by transmission electron microscopy following rotary shadowing. In contrast, the avian Laminin-2 and Laminin-4 isoforms exhibit the characteristic cruciform shape described previously for their mammalian counterparts. Isolated neural crest cells differentially attached and migrated on these laminin isoforms, showing a clear preference for Laminin-G. Similarly to the EHS Laminin-1, neural crest cells recognized all avian isoforms through their alpha 1 beta 1 integrin, shown previously to be the primary laminin-binding receptor on these cells. Neural crest cell interaction with the avian laminins was dependent upon maintenance of the secondary and tertiary structure of the molecules, as shown by the marked reduction in cell attachment and migration upon disruption of the alpha-helical coiled-coil structure of their constituent chains. The results demonstrate that different laminin isoforms may be differentially involved in the regulation of neural crest cell migration and suggest that this regulation operates through interaction of the cells with a structurally conserved cell binding site recognized by the alpha 1 beta 1 integrin.

Animals↗