Isolation and characterization of a laminin-binding protein from rat and chick muscle

A major laminin-binding protein (LBP), distinct from previously described LBPs, has been isolated from chick and rat skeletal muscle (Mr 56,000 and 66,000, respectively). The purified LBPs from the two species were shown to be related antigenically and to have similar NH2-terminal amino acid sequences and total amino acid compositions. Protein blots using laminin and laminin fragments provided evidence that this LBP interacts with the major heparin-binding domain, E3, of laminin. Studies on the association of this LBP with muscle membrane fractions and reconstituted lipid vesicles indicate that this protein can interact with lipid bilayers and has properties of a peripheral, not an integral membrane protein. These properties are consistent with its amino acid sequence, determined from cDNAs (Clegg et al., 1988). Examination by light and electron microscopy of the LBP antigen distribution in skeletal muscle indicated that the protein is localized primarily extracellularly, near the extracellular matrix and myotube plasmalemma. While a form of this LBP has been identified in heart muscle, it is present at low or undetectable levels in other tissues examined by immunocytochemistry indicating that it is probably a muscle-specific protein. As this protein is localized extracellularly and can bind to both membranes and laminin, it may mediate myotube interactions with the extracellular matrix.     

Membrane orientation of laminin binding protein.

Earlier we presented several lines of evidence that a 67-kDa laminin binding protein (LBP) in Leishmania donovani, that is different from the putative mammalian 67-kDa laminin receptor, may play an important role in the onset of leishmaniasis, as these parasites invade macrophages in various organs after migrating through the extracellular matrix. Here we describe the membrane orientation of this Leishmania laminin receptor. Flow cytometric analysis using anti-LBP Ig revealed its surface localization, which was further confirmed by enzymatic radiolabeling of Leishmania surface proteins, autoradiography and Western blotting. Efficient incorporation of LBP into artificial lipid bilayer, as well as its presence in the detergent phase after Triton X-114 membrane extraction, suggests that it may be an integral membrane protein. Limited trypsinization of intact parasite and subsequent immunoblotting of trypsin released material using laminin as primary probe revealed that a major part of this protein harbouring the laminin binding site is oriented extracellularly. Carboxypeptidase Y treatment of the whole cell, as well as the membrane preparation, revealed that a small part of the C-terminal is located in the cytosol. A 34-kDa transmembrane part of LBP could be identified using the photoactive probe, 3-(trifluoromethyl)-3-(m-iodophenyl)diazirine (TID). Partial sequence comparison of the intact protein to that with the trypsin-released fragment indicated that N-terminal may be located extracellularly. Together, these results suggest that LBP may be an integral membrane protein, having significant portion of N-terminal end as well as the laminin binding site oriented extracellularly, a membrane spanning domain and a C-terminal cytosolic end.     

Structure and function of an early divergent form of laminin in hydra: a structurally conserved ECM component that is essential for epithelial morphogenesis

As a major component of the extracellular matrix (ECM), laminin has been found in many vertebrate and invertebrate organisms. Its molecular structure is very similar across species lines and its biological function in the ECM has been extensively studied. In an effort to study ECM structure and function in hydra, we have cloned a partial hydra laminin alpha chain and the full-length hydra laminin beta chain using ECM-enriched cDNA libraries. Analysis of deduced amino acid sequences indicated that both polypeptides have high sequence similarity to a number of invertebrate and vertebrate laminin alpha and beta subunits. Rotary shadow analysis of isolated hydra laminin indicates it has a heterotrimeric organization that is characteristic of vertebrate laminins. A putative integrin-class protein was also identified using a cell-binding peptide sequence from the laminin beta chain as an affinity probe, indicating that integrins are possible cell surface receptors in hydra. In agreement with previous results for the hydra laminin beta chain, in situ hybridization experiments revealed that hydra laminin alpha chain mRNA is restricted to endodermal cells. As with a number of other hydra ECM components, higher levels of laminin alpha chain mRNA are localized to regions where cell migration and differentiation are actively undertaken such as the base of tentacles, the peduncle region, buds, regenerating tentacles, and at the head end during regeneration. The role of laminin in morphogenesis was studied using an antisense approach and the results indicated that translation of the laminin alpha chain is required for head regeneration.     

High affinity binding between laminin and laminin binding protein of Leishmania is stimulated by zinc and may involve laminin zinc-finger like sequences.

In the course of trying to understand the pathogenesis of leishmaniasis in relation to extracellular matrix (ECM) elements, laminin, a major ECM protein, has been found to bind saturably and with high affinity to a 67-kDa cell surface protein of Leishmania donovani. This interaction involves a single class of binding sites, which are ionic in nature, conformation-dependent and possibly involves sulfhydryls. Binding activity was significantly enhanced by Zn2+, an effect possibly mediated through Cys-rich zinc finger-like sequences on laminin. Inhibition studies with monoclonals against polypeptide chains and specific peptides with adhesive properties revealed that the binding site was localized in one of the nested zinc finger consensus sequences of B1 chain containing the specific pentapeptide sequence, YIGSR. Furthermore, incubation of L. donovani promastigotes with C(YIGSR)3-NH2 peptide amide or antibody directed against the 67-kDa laminin-binding protein (LBP) induced tyrosine phosphorylation of proteins with a molecular mass ranging from 115 to 130 kDa. These studies suggest a role for LBP in the interaction of parasites with ECM elements, which may mediate one or more downstream signalling events necessary for establishment of infection.     

Site-restricted expression of cytotactin during development of the chicken embryo

The sequential appearance of the extracellular matrix (ECM) protein, cytotactin, was examined during development of the chicken embryo by immunohistochemical techniques. Although cytotactin was identified as a molecule that mediates glia-neuron interactions, preliminary immunohistochemical localization of the molecule suggested that it was an ECM protein with a widespread but nonetheless more restricted distribution than either fibronectin or laminin. In the present study, it was found that cytotactin is first present in the gastrulating chicken embryo. It appears later in the basement membrane of the developing neural tube and notochord in a temporal sequence beginning in the cephalic regions and proceeding caudally. Between 2 and 3 d of development, the molecule is present at high levels in the early neural crest pathways (surrounding the neural tube and somites) but, in contrast to fibronectin and laminin, is not found in the lateral plate mesoderm or ectoderm. At later times, cytotactin is expressed extensively in the central nervous system, in lesser amounts in the peripheral nervous system, and in a number of nonneural sites, most prominently in all smooth muscles and in basement membranes of lung and kidney. Cytotactin appears in adult tissues with distributions that are similar to those seen in embryonic tissues. The findings raise the possibility that certain ECM proteins contribute to pattern formation in embryogenesis as a result of their restricted expression in a spatiotemporally regulated fashion at some sites but not at others.     

Calsequestrin, an intracellular calcium-binding protein of skeletal muscle sarcoplasmic reticulum, is homologous to aspartactin, a putative laminin-binding protein of the extracellular matrix

Calsequestrin was isolated from chicken fast-twitch skeletal muscle, and partial amino terminal sequence was determined. The sequence (NH2) EEGLNFPTYDGKDRVIDLNE shows high identity with known mammalian calsequestrins contained in the Protein Identification Resource data bank (1). Most importantly, this 20 amino acid sequence shares complete identity with the amino terminus of aspartactin, a putative laminin-binding protein of the extracellular matrix (2, 3). The possible relationship of aspartactin to calsequestrin is discussed.     

Identification and immunolocalization of the laminin binding protein from embryonic avian corneal epithelial cells

We report the identification of a 65-kDa laminin-binding protein (LBP) on the basal cell surface of embryonic corneal epithelium in chicken. The 65-kDa LBP was isolated by affinity chromatography with laminin-Sepharose. When reconstituted with lipid vesicles, it demonstrated specific binding for laminin. We produced monoclonal antibodies (MAbs) against 65-k Da LBP; these MAbs immunohistochemically localized to the basal epithelial cell surface. One MAb interfered with the binding of laminin to isolated epithelia and purified 65-k Da LBP. It appears that we have identified, at least in part, a cell-surface binding site for laminin. This site would provide the important link between the extracellular laminin and the intracellular cytoskeleton, and potentially the metabolic machinery of the corneal epithelial cell.     

Role of 67 kDa cell surface laminin binding protein of Leishmania donovani in pathogenesis.

The role that interaction with laminin may play in Leishmania donovani infection was investigated. Binding of (125)I-radiolabeled laminin, in a liquid-phase assay, by the parasite was rapid, saturable, specific, reversible, and of high affinity. Using a Western blotting procedure, a 67 kDa laminin-binding protein (LBP) was identified from the membrane of both the promastigote and amastigote forms of L. donovani. Subsequently, the protein was purified by affinity chromatography. Immunofluorescence with a polyclonal antibody against LBP as well as flow cytometric analysis demonstrated its presence at the parasite surface. After stimulation with phorbol-12-myristate-13-acetate (PMA), U937 cells exhibited the ability to adhere to laminin and LBP specifically inhibited this adhesion. The reduced parasite adhesion after tunicamycin treatment suggested the importance of sugar residues in cell adhesion. Although co-administration of either laminin or LBP or anti LBP antibody reduced parasite virulence, resulting in a lower level of infection in the BALB/c mouse model, an in vitro macrophage culture-enhanced level of infection was observed in the case of laminin-coated parasites. The results collectively suggest a role for LBP in the interaction of the parasite with extracellular matrix elements, which may constitute a basis for the homing of the parasite to its physiological address.     

Induction of extracellular matrix gene expression in normal human keratinocytes by transforming growth factor beta is altered by cellular differentiation

Changes in epithelial substrate have been related to the cellular capacity for proliferation and to changes in cellular behavior. The effect of TGF beta 1 on the expression of the basement membrane genes, fibronectin, laminin B1, and collagen alpha 1 (IV), was examined. Northern analysis revealed that treatment of normal human epidermal keratinocytes with 100 pM TGF beta 1 increased the expression of each extracellular matrix (ECM) gene within 4 h of treatment. Maximal induction was reached within 24 h after treatment. The induction of ECM mRNA expression was dose dependent and was observed at doses as low as 1-3 pM TGF beta 1. Incremental doses of TGF beta 1 also increased cellular levels of fibronectin protein in undifferentiated keratinocytes and resulted in increased secretion of fibronectin. Squamous-differentiated cultures of keratinocytes expressed lower levels of the extracellular matrix RNAs than did undifferentiated cells. Treatment of these differentiated cells with TGF beta 1 induced the expression of fibronectin mRNA to levels seen in TGF beta-treated, undifferentiated keratinocytes but only marginally increased the expression of collagen alpha 1 (IV) and laminin B1 mRNA. The increased fibronectin mRNA expression in the differentiated keratinocytes was also reflected by increased accumulation of cellular and secreted fibronectin protein. The inclusion of cycloheximide in the protocol indicated that TGF beta induction of collagen alpha 1 (IV) mRNA was signaled by proteins already present in the cells but that TGF beta required the synthesis of a protein(s) to fully induce expression of fibronectin and laminin B1 mRNA. The differential regulation of these genes in differentiated cells may be important to TGF beta action in regulating reepithelialization.     

Ribosome-associated protein LBP/p40 binds to S21 protein of 40S ribosome: analysis using a yeast two-hybrid system

The ribosome-associated protein LBP/p40, which was originally named after "laminin binding protein precursor p40," is distributed on the cell surface as laminin binding protein p67 (LBP/p67), in the nucleus, and on 40S ribosomes. In a broad range of eukaryotes, the localization of LBP/p40 on the 40S ribosome is well conserved. Two yeast homologs of LBP/p40 are believed to be essential for cell viability and each gene product probably corresponds to the assembly and/or stability of the 40S ribosomal subunit. The precise role of LBP/p40 in translation, however, remains to be elucidated, especially in higher eukaryotes. In this report, we used a yeast two-hybrid screening method to isolate molecules associated with human LBP/p40 protein on ribosomes. We found that the 40S ribosomal protein S21 was tightly bound with LBP/p40 in this yeast two-hybrid system and in in vitro analysis. Further, we discovered that the association required a broad region of the LBP/p40 amino acid sequence, which corresponds to the highly conserved region of LBP/p40 homologs among eukaryotes.     

Laminins in Peripheral Nerve Development and Muscular Dystrophy

Laminins are extracellular matrix (ECM) proteins that play an important role in cellular function and tissue morphogenesis. In the peripheral nervous system (PNS), laminins are expressed in Schwann cells and participate in their development. Mutations in laminin subunits expressed in the PNS and in skeleton muscle may cause peripheral neuropathies and muscular dystrophy in both humans and mice. Recent studies using gene knockout technology, such as cell-type specific gene targeting techniques, revealed that laminins and their receptors mediate Schwann cell and axon interactions. Schwann cells with disrupted laminin expression exhibit impaired proliferation and differentiation and also undergo apoptosis. In this review, we focus on the potential molecular mechanisms by which laminins participate in the development of Schwann cells.     

Primary structure of locust flight muscle fatty acid binding protein.

The amino acid sequence of the fatty acid binding protein (FABP) from flight muscle of the locust, Schistocerca gregaria, has been determined. The sequence of the N-terminal 39 amino acid residues, determined by automated Edman degradation, was used to prepare a degenerate oligonucleotide that corresponded to amino acid residues 16-23. cDNA coding for FABP was constructed from flight muscle mRNA and amplified by the polymerase chain reaction using the degenerate oligonucleotide and an oligo dT-NotI primer adapter as primers. The amplification product was cloned and sequenced. Additionally, a cDNA library of flight muscle mRNA was prepared and screened with a 414-bp probe prepared from the clone. The primary structure of locust FABP was compared with the proteins in the Swiss protein databank and found to have significant homology with mammalian FABPs over the entire 133-residue sequence. The best match was versus human heart FABP (41% identity), attesting to the highly conserved nature of this protein. The results suggest that locust muscle FABP is a member of the lipid binding protein superfamily and may provide valuable insight into the evolution of this abundant protein class.     

Glyceraldehyde 3-phosphate dehydrogenase protein and mRNA are both differentially expressed in adult chickens but not chick embryos

We have determined the 679 nucleotide sequence of a cDNA clone which, by hybridization-translation experiments, corresponds to a 36K chick brain protein. Our studies provide a partial amino acid sequence for this protein, identifying it as chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Antisera raised against purified chicken GAPDH reacted with a 36K protein present in chick brain extracts and estimated to be the fourth most prevalent protein, as determined by either Coomassie Blue staining or by in vitro translation of chick brain mRNA. The amounts of GAPDH mRNA in chick brain, liver and muscle and adult chicken brain are similar, whereas the relative amount of adult chicken muscle GPDH mRNA is greatly elevated and that of adult liver lowered. The GAPDH protein levels showed a similar variation between tissues, suggesting that the levels of GAPDH protein are largely regulated by the amount of available GAPDH mRNA. The chicken GAPDH clone does not hybridize to rat mRNA, even though GAPDH is one of the most evolutionarily conserved proteins, indicating that selection pressures are heavier at the primary protein sequence level than at the nucleic acid sequence level for this gene, a situation contrasting to that of the tubulins.     

Enzymatic activity and in vivo distribution of 5'-nucleotidase, an extracellular matrix binding glycoprotein, during the development of chicken striated muscle.

The ecto-enzyme 5'-nucleotidase isolated from chicken gizzard has previously been shown to be a potent ligand of two glycoproteins of the extracellular matrix, namely fibronectin and laminin. Using immunofluorescent labeling techniques we observed that 5'-nucleotidase codistributed with laminin during the development of chicken striated muscle. In contrast, ecto-5'-nucleotidase was only faintly detectable on cells surrounded by a matrix expressing high levels of fibronectin. This distribution pattern distinguished 5'-nucleotidase from the pluripotent extracellular matrix receptors, chicken beta 1-integrins, which are expressed equally well in muscle and connective tissue. In addition, the specific activity of striated muscle ecto-5'-nucleotidase was stable during development and increased markedly posthatching. At each age considered, this specific activity corresponded to an 80-kDa enzyme which was inhibited by alpha,beta-methyleneadenosine diphosphate or by a monoclonal antibody directed against the smooth muscle isoform of the enzyme. Previous in vitro studies have revealed that 5'-nucleotidase is involved in the spreading of various mesenchyme-derived cells, such as chicken embryonic fibroblasts and myoblasts, on a laminin substrate. A prerequisite to examining a potential in vivo role for 5'-nucleotidase as an extracellular matrix ligand was to study its distribution. In adult muscle, 5'-nucleotidase displayed a more restricted distribution than in embryo. Results show that, in vivo, 5'-nucleotidase is revealed by immunofluorescent labeling using poly- and monoclonal antibodies to chicken gizzard 5'-nucleotidase in two structures, the costameres and myotendinous junctions, which are closely related to the focal adhesion sites observed in cell culture.     

Ontogenesis of the murine hepatic extracellular matrix: an immunohistochemical study

To define the role of the extracellular matrix (ECM) in hepatogenesis, we examined the temporal and spatial deposition of fibronectin, laminin and collagen types I and IV in 12.5-21.5 day fetal and 1, 7 and 14 day postnatal rat livers. In early fetal liver, discontinuous deposits of the four ECM components studied were present in the perisinusoidal space, with laminin being the most prevalent. All basement membrane zones contained collagen type IV and laminin, including those of the capsule (mesothelial), portal vein radicles and bile ductules. Fibronectin had a distribution similar to that of collagen type IV early in gestation. However, at later gestational dates, fibronectin distribution in the portal triads approached that of collagen type I, being present in the interstitial connective tissues; whereas, collagen type IV and laminin were restricted to vascular and biliary basement membrane zones in those regions. The cytoplasm of some sinusoidal lining cells and hepatocytes reacted with antibodies to extracellular matrix components. By electron microscopy the immunoreactive material was localized in the endoplasmic reticulum, indicating the ability of these cells to synthesize these ECM proteins. Biliary ductular cells had prominent intracytoplasmic staining for laminin and collagen type IV from day 19.5 gestation until 7 days of postnatal life, but lacked demonstrable fibronectin or collagen type I. These results demonstrate that by 12.5 days of gestation the rat liver anlage has deposited a complex extracellular matrix in the perisinusoidal space. The prevalence of laminin in the developing hepatic lobules suggests a possible role for this glycoprotein in hepatic morphogenesis. In view of the intimate association of the hepatic lobular extracellular matrix with the developing vasculature, we hypothesize that laminin provides a scaffold of the developing liver, but once the ontogenesis is complete, intrahepatic perisinusoidal laminin expression is suppressed.     

Regulation of rat mammary gene expression by extracellular matrix components

In the mammary gland the induction and maintenance of differentiation are dependent on both lactogenic hormones and the extracellular matrix (ECM). Since mammary epithelial cells differentiate on a basement membrane in vivo we have examined the effects of basement membrane components on the expression of milk protein genes in primary rat mammary cultures. We examined the effects of a basement membrane gel derived from the Englebreth-Holm-Swarm tumor as well as its major component, laminin, on the expression of a group of milk protein genes. We demonstrate that the basement membrane gel induces alpha-casein and alpha-lactalbumin (alpha-LA) accumulation up to 160- and 70-fold, respectively, of that on tissue culture plastic. Laminin, a major component of the basement membrane, also caused significant induction of these same proteins. In order to determine whether these ECM effects occurred at a translational or post-translational level, pulse-chase experiments were performed. These experiments demonstrated that a laminin substratum selectively effects milk protein turnover and secretion. In order to demonstrate whether ECM effects occurred at the level of steady state accumulation of mRNA we performed dot blot and Northern analyses using cloned cDNA probes for alpha-, beta-, and gamma-caseins and alpha-LA. These studies demonstrated that ECM components induced alpha- and beta-caseins up to 10-fold, and alpha-LA up to 3-fold, with no significant effect on gamma-casein. These results demonstrate that milk protein genes are not coordinately regulated by ECM components. Furthermore, since the amount of induction of milk proteins exceeds the amount of induction of mRNAs for these proteins, we conclude that in our system a major effect of ECM components is at the translational and/or post-translational levels. Based on these findings we propose a model in which basement membrane components effect mammary gene expression at multiple levels.     

Nucleotide sequence of the gene encoding respiratory syncytial virus matrix protein.

The amino acid sequence of the matrix protein of the human respiratory syncytial virus (RS virus) was deduced from the sequence of a cDNA insert in a recombinant plasmid harboring an almost full-length copy of this gene. It specifically hybridized to a single 1,050-base mRNA from infected cells. The recombinant containing 944 base pairs of RS viral matrix protein gene sequence lacked five nucleotides corresponding to the 5' end of the mRNA. The nucleotide sequence of the 5' end of the mRNA was determined by the dideoxy sequencing method and found to be 5' NGGGC, wherein the C residue is one nucleotide upstream of the cloned viral sequence. The initiator ATG codon for the matrix protein is embedded in an AATATGG sequence similar to the canonical PXXATGG sequence present around functional eucaryotic translation initiation codons. There is no conserved sequence upstream of the polyadenylate tail, unlike vesicular stomatitis virus and Sendai virus, in which four nucleotides upstream of the polyadenylate tail are conserved in all genes. There is no equivalent of the eucaryotic polyadenylation signal AAUAAA upstream of the polyadenylate tail. The matrix protein of 28,717 daltons has 256 amino acids. It is relatively basic and moderately hydrophobic. There are two clusters of hydrophobic amino acid residues in the C-terminal third of the protein that could potentially interact with the membrane components of the infected cell. The matrix protein has no homology with the matrix proteins of other negative-strand RNA viruses, implying that RS virus has undergone extensive evolutionary divergence. A second open reading frame potentially encoding a protein of 75 amino acids and partially overlapping the C terminus of the matrix protein was also identified.