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P D Yurchenco

Publications and source records attributed to P D Yurchenco.

At least 19 recordsLinked to original sources
PubMed1997

Self-assembly of laminin isoforms.

The alpha, beta, and gamma subunits of basement membrane laminins can combine into different heterotrimeric molecules with either three full short arms (e.g. laminins-1-4), or molecules containing one (laminins-6-9) or more (laminin-5) short arm truncations. Laminin-1 (alpha1beta1gamma1), self-assembles through a calcium-dependent thermal gelation requiring binding interactions between N-terminal short arm domains, forming a meshwork polymer thought to contribute to basement membrane architecture (Yurchenco, P. D., and Cheng, Y. S. (1993) J. Biol. Chem. 268, 17286-17299). However, it has been unclear whether other isoforms share this property, and if so, which ones. To begin to address this, we evaluated laminin-2 (alpha2beta1gamma1), laminin-4 (alpha2beta2gamma1), laminin-5 (alpha3Abeta3gamma2), and laminin-6 (alpha3Abeta1gamma1). The first two isoforms were found to self-aggregate in a concentration- and temperature-dependent manner and a close self-assembly relationship among laminins-1, -2, and -4 were demonstrated by: (a) polymerization of all three proteins was inhibited by EDTA and laminin-1 short arm fragments, (b) polymerization of laminin-1 was inhibited by fragments of laminins-2 and -4, (c) laminin-2 and, to a lesser degree, laminin-4, even well below their own critical concentration, co-aggregated with laminin-1, evidence for co-polymerization. Laminin-5, on the other hand, neither polymerized nor co-polymerized with laminin-1. Laminin-6 failed to co-aggregate with laminin-1 at all concentrations evaluated, evidence for a lack of a related self-assembly activity. The data support the hypothesis that all three short arms are required for self-assembly and suggest that the short arm domain structure of laminin isoforms affect their architecture-forming properties in basement membranes.

Animals↗
PubMed1997

Localization of heparin binding activity in recombinant laminin G domain.

Basement membrane laminin (laminin-1) is a multidomain glycoprotein that interacts with itself, heparin and cells. The interaction with heparin/heparan sulfate proteglycans is thought to be important for the architectural formation of basement membranes and adhesion to cells. The major heparin binding site has been known to reside in the long arm globular domain (G domain). The G domain is in turn subdivided into five subdomains (G1-G5). In order to localize the heparin binding regions further, recombinant G domains (rG and rG5) were expressed in Sf9 insect cells using baculovirus expression vector. By the limited proteolysis of recombinant G domains followed by either heparin affinity HPLC or overlay with radiolabeled heparin, the relative affinity of each subdomain to heparin was assigned as G1>G2 = G4>G5>G3, such that G1 bound strongly and G3 not at all. Since the activity in G1-G3 is cryptic in intact laminin long arm [Sung, U., O'Rear, J. J. & Yurchenco, P. D. (1993) J. Cell Biol. 123, 1255-1268], the active heparin binding site of G domain appears to be located in G4 and proximal G5.

Amino Acid Sequence↗
PubMed1997

The laminin alpha2-chain short arm mediates cell adhesion through both the alpha1beta1 and alpha2beta1 integrins.

Laminin-2, a heterotrimer composed of alpha2, beta1, and gamma1 subunits, is the primary laminin isoform found in muscle and peripheral nerve and is essential for the development and stability of basement membranes in these tissues. Expression of a domain VI-truncated laminin alpha2-chain results in muscle degeneration and peripheral nerve dysmyelination in the dy2J dystrophic mouse. We have expressed amino-terminal domains VI through IVb of the laminin alpha2-chain, as well as its laminin-1 alpha1-chain counterpart, to identify candidate cell-interactive functions of this critical region. Using integrin-specific antibodies, recognition sites for the alpha1beta1 and alpha2beta1 integrins were identified in the short arms of both laminin alpha1- and alpha2-chain isoforms. Comparisons with a beta-alpha chimeric short arm protein possessing beta1-chain domain VI further localized these activities to alpha-chain domain VI. In addition, we found that the laminin alpha2-chain short arm supported neurite outgrowth independent of other laminin-2 subunits. A heparin/heparan sulfate binding activity was also localized to this region of the laminin alpha2 subunit. These data provide the first evidence that domain VI of the laminin alpha2-chain mediates interactions with cell surface receptors and suggest that these integrin and heparin binding sites, alone or in concert, may play an important role in muscle and peripheral nerve function.

Animals↗
PubMed1997

The alpha chain of laminin-1 is independently secreted and drives secretion of its beta- and gamma-chain partners.

A mammalian recombinant strategy was established to dissect rules of basement membrane laminin assembly and secretion. The alpha-, beta-, and gamma-chain subunits of laminin-1 were expressed in all combinations, transiently and/or stably, in a near-null background. In the absence of its normal partners, the alpha chain was secreted as intact protein and protein that had been cleaved in the coiled-coil domain. In contrast, the beta and gamma chains, expressed separately or together, remained intracellular with formation of betabeta or betagamma, but not gammagamma, disulfide-linked dimers. Secretion of the beta and gamma chains required simultaneous expression of all three chains and their assembly into alphabetagamma heterotrimers. Epitope-tagged recombinant alpha subunit and recombinant laminin were affinity-purified from the conditioned medium of alphagamma and alphabetagamma clones. Rotary-shadow electron microscopy revealed that the free alpha subunit is a linear structure containing N-terminal and included globules with a foreshortened long arm, while the trimeric species has the typical four-arm morphology of native laminin. We conclude that the alpha chain can be delivered to the extracellular environment as a single subunit, whereas the beta and gamma chains cannot, and that the alpha chain drives the secretion of the trimeric molecule. Such an alpha-chain-dependent mechanism could allow for the regulation of laminin export into a nascent basement membrane, and might serve an important role in controlling basement membrane formation.

Basement Membrane↗
PubMed1997

Neural targeting of Mycobacterium leprae mediated by the G domain of the laminin-alpha2 chain.

We report that the molecular basis of the neural tropism of Mycobacterium leprae is attributable to the specific binding of M. leprae to the laminin-alpha2 (LN-alpha2) chain on Schwann cell-axon units. Using recombinant fragments of LN-alpha2 (rLN-alpha2), the M. leprae-binding site was localized to the G domain. rLN-alpha2G mediated M. leprae binding to cell lines and to sciatic nerves of dystrophic dy/dy mice lacking LN-alpha2, but expressing laminin receptors. Anti-beta4 integrin antibody attenuated rLN-alpha2G-mediated M. leprae adherence, suggesting that M. leprae interacts with cells by binding to beta4 integrin via an LN-alpha2G bridge. Our results indicate a novel role for the G domain of LN-2 in infection and reveal a model in which a host-derived bridging molecule determines nerve tropism of a pathogen.

Animals↗
PubMed1997

Laminin-induced clustering of dystroglycan on embryonic muscle cells: comparison with agrin-induced clustering.

The effect of laminin on the distribution of dystroglycan (DG) and other surface proteins was examined by fluorescent staining in cultures of muscle cells derived from Xenopus embryos. Western blotting confirmed that previously characterized antibodies are reactive in Xenopus. In control cultures, alphaDG, betaDG, and laminin binding sites were distributed as microclusters (<1 microm2 in area) over the entire dorsal surface of the muscle cells. Treatment with laminin induced the formation of macroclusters (1-20 microm2), accompanied by a corresponding decline in the density of the microclusters. With 6 nM laminin, clustering was apparent within 150 min and near maximal within 1 d. Laminin was effective at 30 pM, the lowest concentration tested. The laminin fragment E3, which competes with laminin for binding to alphaDG, inhibited laminin-induced clustering but did not itself cluster DG, thereby indicating that other portions of the laminin molecule in addition to its alphaDG binding domain are required for its clustering activity. Laminin-induced clusters also contained dystrophin, but unlike agrin-induced clusters, they did not contain acetylcholine receptors, utrophin, or phosphotyrosine, and their formation was not inhibited by a tyrosine kinase inhibitor. The results reinforce the notion that unclustered DG is mobile on the surface of embryonic muscle cells and suggest that this mobile DG can be trapped by at least two different sets of molecular interactions. Laminin self binding may be the basis for the laminin-induced clustering.

Agrin↗
PubMed1997

Developmental expression of perlecan during murine embryogenesis.

Perlecan is a modular heparan sulfate proteoglycan that is an intrinsic constituent of all basement membranes and extracellular matrices. Because of its strategic position and unique structure, perlecan has been implicated in modulating the activity of various growth factors required for normal development and tissue homeostasis. To gain insights into the potential function of perlecan in vivo, we examined the spatiotemporal distribution of its mRNA and protein core during murine embryogenesis. We utilized a new affinity-purified antibody that recognizes specifically the protein core of perlecan together with an in situ RT-PCR approach to perform a systematic analysis of perlecan expression and deposition during murine ontogeny. Perlecan appeared early (E10.5) in tissues of vasculogenesis including heart, pericardium, and major blood vessels. Its early expression coincided with the development of the cardiovascular system. Subsequently (E11-13), the greatest deposition of perlecan occurred within the developing cartilage, especially the cartilage undergoing endochondral ossification, where it remained elevated throughout all the developmental stages, and up to adulthood. Interestingly, the mRNA levels of perlecan were always higher in all the vascularized tissues, principally within endothelial cells, while chondrocytes displayed relatively low mRNA levels. This suggests a higher biosynthesis and turnover rates in the blood vessels vis-à-vis those of cartilaginous and other mesenchymal tissues. During later stages of development (E13-17.5) perlecan mRNA levels progressively increased and its expression correlated with the onset of tissue differentiation of various parenchymal organs including the developing kidneys, lungs, liver, spleen, and gastrointestinal tract. The central nervous system showed no perlecan expression with the exception of the calvaria and choroid plexus. Collectively, the results indicate that perlecan may play crucial roles not only in vasculogenesis but also in the maturation and maintenance of differentiated tissues, including cartilage.

Animals↗
PubMed1997

Mild congenital muscular dystrophy in two patients with an internally deleted laminin alpha2-chain.

Congenital muscular dystrophy (CMD) is a group of clinically and genetically heterogeneous disorders inherited in an autosomal recessive mode. The alpha2-chain of laminin-2 (previously called merosin) has been shown by immunohistochemical and genetic analyses to be implicated in the pathogenesis of the 'classic' form of CMD. In the 'merosin-deficient' subgroup, which represents about half of the cases, more definite evidence of the involvement of the laminin alpha2-chain has recently been reported with the identification of mutations in the gene encoding the alpha2-chain of laminin 2 (LAMA2) in CMD patients. Here we report on two siblings from a consanguineous family expressing an internally deleted laminin alpha2-chain as a result of a splice site mutation in the LAMA2 gene which causes the splicing of exon 25. The predicted protein lacks 63 amino acids in domain IVa which forms a globular structure on the short arm of the alpha2-chain. Interestingly, these patients appear mildly affected compared to others who completely lack this protein. This situation presents a striking analogy with Becker muscular dystrophy, where in-frame deletions in the dystrophin gene result in the expression of a semi-functional protein and lead to a mild phenotype.

Amino Acid Sequence↗
PubMed1995

Mapping of network-forming, heparin-binding, and alpha 1 beta 1 integrin-recognition sites within the alpha-chain short arm of laminin-1.

Cell-interactive and architecture-forming functions are associated with the short arms of basement membrane laminin-1. To map and characterize these functions, we expressed recombinant mouse laminin-1 alpha-chain extending from the N terminus through one third of domain IIIb. This dumbbell-shaped glycoprotein (r alpha 1(VI-IVb)'), secreted by mammalian cells, was found to possess three activities. 1) Laminin polymerization was quantitatively inhibited by recombinant protein, supporting an alpha-chain role for a three-short arm interaction model of laminin self-assembly. 2) r alpha 1(VI-IVb)' bound to heparin, and the activity was localized to a subfragment corresponding to domain VI by 125I-heparin blotting. 3) PC12 rat pheochromocytoma cells adhered to, and rapidly extended branching neurites on, r alpha 1(VI-IVb)', with adhesion inhibited by alpha 1 and beta 1 integrin chain-specific antibodies. The ability of anti-laminin antibody to block PC12 cell adhesion to laminin was selectively prevented by absorption with r alpha 1(VI-IVb)' or alpha-chain domain VI fragment. This active integrin-recognition site could furthermore be distinguished from a second cryptic alpha 1 beta 1-binding site exposed by heat treatment of fragment P1', a short arm fragment lacking globules. Thus, a polymer-forming, a heparin-binding, and the active alpha 1 beta 1 integrin-recognition site are all clustered at the end of the alpha-chain short arm, the latter two resident solely in domain VI.

Amino Acid Sequence↗
PubMed1994

High resolution platinum-carbon replication of freeze-dried basement membrane.

High angle platinum/carbon (Pt/C) replication has proved to be a valuable tool in analyzing basement membrane structure in human amnion, bovine lens capsule, and the Engelbreth-Holm-Swarm (EHS) tumor. High resolution replicas for transmission electron microscopy (TEM) have been achieved by depositing 1.0 +/- 0.1 nm thick Pt/C films backed with rotary deposited 12.5 +/- 2.5 nm thick carbon films. The basement membrane collagen IV network was observed to consist of fine branching filaments containing globular domains intrinsic to the filaments. A second quasi-regular network is formed by laminin. Unidirectional 45 degree angle Pt/C replication was used for most of this work. The merits and deficiencies of unidirectional vertical replication (80 degree angle), unidirectional 45 degree angle, and 20 degrees low angle rotary replication are discussed. Vertical replication produces the highest resolution replicas and has the potential for revealing the overall pattern of basement membrane structural assembly if basement membrane preparations freeze-dried in low salt can faithfully maintain their in vivo structure.

Amnion↗
PubMed1994

Domain-specific activation of neuronal migration and neurite outgrowth-promoting activities of laminin.

The ECM glycoprotein laminin has profound and varied actions on neurons in vitro. Little is known about how laminin's multiple domains and receptor-binding sites interact in determining its overall effects. Here, it is shown that laminin's ability to promote migration of olfactory epithelium neuronal cells maps to distal long arm domain E8 and is mediated by alpha 6 beta 1 integrin. Surprisingly, treatment of laminin with antibodies against its short arms (domains E1' or P1') uncovered a new neuronal migration-promoting activity, mediated by a beta 1 integrin other than alpha 6 beta 1. Laminin treated with anti-short arm antibodies also promoted beta 1 integrin-dependent neurite outgrowth from late embryonic retinal neurons, which are normally unresponsive to laminin. These "antibody-induced" migration and neurite outgrowth activities mapped to laminin's distal long arm, far from the site(s) of antibody binding. Evidence is presented that the induced activities are not actually cryptic in laminin, but are suppressed by an activity that is located in laminin's P1' domain and that may be lacking in the laminin homolog merosin.

Animals↗
PubMed1994

Basal lamina assembly.

From studies of the 'classical' components, models for the assembly and structure of an idealized basal lamina have been developed. In particular, the evidence supports the concept of enmeshed collagen and laminin polymers, in which nidogen/entactin acts as a bridge between these molecules and provides anchorage for diverse matrix components. Different basement membranes, however, possess different members of the basic basal lamina families, such as the newly described alpha 6 (IV) collagen, alpha 2 (merosin) laminin, and beta 3 laminin (in kalinin/nicein) chains. Even though these members share homologous domains and sequences, and are likely to share certain functions, they also possess unique characteristics that are expected to provide for basal lamina heterogeneity. A combination of genetic, recombinant and biochemical approaches are now being applied to elucidate the special roles of both old and new components.

Animals↗
PubMed1994

Ductus arteriosus smooth muscle cell migration on collagen: dependence on laminin and its receptors.

During permanent closure of the ductus arteriosus, smooth muscle cells migrate through the extracellular matrix (ECM) to form intimal mounds that occlude the vessel's lumen. Smooth muscle cells (SMC) migrate over surfaces coated with collagen in vitro. During the migration SMC also synthesize fibronectin (FN) and laminin (LN). Antibodies against FN and LN inhibit migration on collagen by 30% and 67%, respectively. Because of the apparent importance of LN in migration, we examined how SMC interact with LN and LN fragments (P1, E8, P1', E1', E3, E4, and G). Ductus SMC adhere to high concentrations of LN and two fragments of the molecule: P1 and E8. They use a unique set of integrin receptors to bind to LN (alpha 1 beta 1, alpha 6 beta 1 and alpha v beta 3), to P1 (alpha 1 beta 1, alpha v beta 3), and to E8 (alpha 6 beta 1, alpha v beta 3). The alpha v beta 3 integrin binds to the P1 fragment of LN in an RGD peptide-dependent manner, and to the E8 fragment in an RGD-independent manner; the RGD site on the P1 fragment probably is not available to the cell in intact LN. Antibodies against beta 1 integrins completely inhibit SMC adhesion to LN; antibodies against the alpha v beta 3 integrin do not block SMC adhesion to LN, but do prevent cell spreading. LN is also capable of interfering with SMC adhesion to other ECM components. The antiadhesive effect of LN is located in the E1' domain. Both exogenous and endogenous LN increase SMC motility on collagen I. The locomotion-promoting activity of LN resides in the E1' antiadhesive domain, and not in its adhesive (P1, E8) domains. LN causes a decrease in the number of focal contacts on collagen I. This might enable SMC to alter their mobility as they move through the extracellular matrix to occlude the ductus arteriosus lumen.

Amino Acid Sequence↗
PubMed1993

Self-assembly and calcium-binding sites in laminin. A three-arm interaction model.

Laminin, a four-arm glycoprotein, polymerizes in vitro into networks similar to those found in basement membranes. We have dissected this calcium-dependent assembly by analyzing proteolytic fragment binding using equilibrium gel filtration, ultracentrifugation, and electron microscopy. The cathepsin G fragment C1-4 (three short arms) was found to aggregate with a critical concentration similar to that for laminin. Like laminin polymerization, this assembly was inhibited by the smaller elastase short arm fragments E4 (B1 domains VI and V) and E1' (A-B2 short arm complex). Fragment E4 bound directly to E1' in a calcium-dependent manner (KD = 1.4 microM). In contrast, homologous self-interactions of short arm fragments and all interactions of long arm fragments were considerably weaker or nonexistent. While electron micrographs of E1' or E4 alone contained mostly monomers, those of E1'/E4 mixtures contained dimers and oligomers with E1' dimers connected to each other through their A and B2 arms, often with visible E4 molecules at their junctions. 45Ca2+ bound principally to fragment E1' with localization to the end of the B2 chain. These data support a model in which polymerization requires the interaction of all three ligands, one from each short arm, with calcium activating assembly by binding to the B2 short arm.

Amino Acid Sequence↗
PubMed1993

Laminin-binding protein 120 from brain is closely related to the dystrophin-associated glycoprotein, dystroglycan, and binds with high affinity to the major heparin binding domain of laminin.

When brain proteins separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose are probe with 125I-labeled laminin, a single broad band of approximately 120 kDa binds laminin specifically. We show here by two-dimensional electrophoresis and protein microsequencing that this band consists of two distinct laminin-binding proteins. One of these is the amyloid precursor protein. The other, laminin-binding protein (LBP) 120, is closely related to the dystrophin-associated glycoprotein, dystroglycan (156 kDa); 5 peptides from purified bovine brain LBP120, ranging in size from 7 to 19 residues, are up to 100% identical to the predicted amino acid sequence of muscle dystroglycan (ibraghimov-Beskrovanaya, O., Ervasti, J. M., Leveille, C. J., Slaughter, C. A., Sernett, S. W., and Campbell, K. P. (1992) Nature 355, 696-702). These protein microsequence data support the data of Ibraghimov-Beskrovnaya et al., which suggest that the dystroglycan precursor is processed into 120/156- and 43-kDa proteins. Moreover, the data suggest a revision in the position of the proposed cleavage site of the precursor. The glycosylation and extracellular localization of LBP120/dystroglycan are consistent with it being a cell surface laminin receptor. LBP120/dystroglycan, either as a native protein, or following SDS-PAGE and transfer to nitrocellulose, binds with high affinity (Kd = 90 nM) to a proteolytic fragment of laminin (E3) containing the major heparin binding domain. This binding is Ca(2+)-dependent and inhibited by low concentrations of heparin. Thus, LBP120/dystroglycan is a major non-integrin laminin receptor whose high affinity interaction with laminin may reflect a structural role in brain and muscle.

Amino Acid Sequence↗
PubMed1993

Recombinant laminin G domain mediates myoblast adhesion and heparin binding.

A recombinant mouse cDNA fragment encoding the G domain of the basement membrane laminin A chain was inserted into the eukaryotic baculovirus expression vector pVL1392 modified to produce fusion proteins carrying the rat fibronectin signal. G domain, expressed and secreted as a soluble glycoprotein (rG), was purified to near homogeneity without denaturing conditions. By electron microscopy rG possessed the same globular morphology as found in laminin. rG was cleaved with elastase into two fragments, rG70 and rG50. The latter fragment possessed the identical N terminus as laminin fragment E3 and both shared the same secondary structure by circular dichroism. rG, and rG containing a deletion (residues 2980-3028) rich in basic residues bound to heparin with similar avidity. rG also promoted mouse C2 myoblast cell adhesion and spreading, and evaluation of myoblasts on rG70 and rG50 further revealed that cell spreading was an activity confined to the more proximal sequence of rG70. Antibody specific for rG70 completely blocked cell adhesion to intact laminin in contrast to antibody specific for E3. Finally rG did not inhibit laminin polymerization. These data support the role of G domain in cell and heparin binding, but not laminin self-assembly, and the approach provides a means to further characterize these functions.

Animals↗