Chisei Shimono

Transcriptome-based systematic identification of extracellular matrix proteins

Extracellular matrix (ECM), which provides critical scaffolds for all adhesive cells, regulates proliferation, differentiation, and apoptosis. Different cell types employ customized ECMs, which are thought to play important roles in the generation of so-called niches that contribute to cell-specific functions. The molecular entities of these customized ECMs, however, have not been elucidated. Here, we describe a strategy for transcriptome-wide identification of ECM proteins based on computational screening of >60,000 full-length mouse cDNAs for secreted proteins, followed by in vitro functional assays. These assays screened the candidate proteins for ECM-assembling activities, interactions with other ECM molecules, modifications with glycosaminoglycans, and cell-adhesive activities, and were then complemented with immunohistochemical analysis. We identified 16 ECM proteins, of which seven were localized in basement membrane (BM) zones. The identification of these previously unknown BM proteins allowed us to construct a body map of BM proteins, which represents the comprehensive immunohistochemistry-based expression profiles of the tissue-specific customization of BMs.

Activin A Binds to Perlecan through Its Pro-region That Has Heparin/Heparan Sulfate Binding Activity

Activin A, a member of the transforming growth factor-β family, plays important roles in hormonal homeostasis and embryogenesis. In this study, we produced recombinant human activin A and examined its abilities to bind to extracellular matrix proteins. Recombinant activin A expressed in 293-F cells was purified as complexes of mature dimeric activin A with its pro-region. Among a panel of extracellular matrix proteins tested, recombinant activin A bound to perlecan and agrin, but not to laminins, nidogens, collagens I and IV, fibronectin, and nephronectin. The binding of recombinant activin A to perlecan was inhibited by heparin and high concentrations of NaCl and abolished by heparitinase treatment of perlecan, suggesting that activin A binds to the heparan sulfate chains of perlecan. In support of this possibility, recombinant activin A was capable of directly binding to heparin and heparan sulfate chains. Site-directed mutagenesis of recombinant activin A revealed that clusters of basic amino acid residues, Lys259-Lys263 and Lys270-Lys272, in the pro-region were required for binding to perlecan. Interestingly, deletion of the peptide segment Lys259-Gly277 containing both basic amino acid clusters from the pro-region did not impair the activity of activin A to stimulate Smad-dependent gene expressions, although it completely ablated the perlecan-binding activity. The binding of activin A to basement membrane heparan sulfate proteoglycans through the basic residues in the pro-region was further confirmed by in situ activin A overlay assays using frozen tissue sections. Taken together, the present results indicate that activin A binds to heparan sulfate proteoglycans through its pro-region and thereby regulates its localization within tissues.

Dual Function of the Propeptide of Prouroguanylin in the Folding of the Mature Peptide DISULFIDE-COUPLED FOLDING AND DIMERIZATION

Guanylyl cyclase activating peptide II (GCAP-II), an endogenous ligand of guanylyl cyclase C, is produced via the processing of the precursor protein (prepro-GCAP-II). We have previously shown that the propeptide in pro-GCAP-II functions as an intramolecular chaperone in the proper folding of the mature peptide, GCAP-II (Hidaka, Y., Ohno, M., Hemmasi, B., Hill, O., Forssmann, W.-G., and Shimonishi, Y. (1998) Biochemistry 37, 8498–8507). Here, we report an essential region in pro-GCAP-II for the correct disulfide pairing of the mature peptide, GCAP-II. Five mutant proteins, in which amino acid residues were sequentially deleted from the N terminus, and three mutant proteins of pro-GCAP-II, in which N-terminal 6, 11, or 17 amino acid residues were deleted, were overproduced usingEscherichia coli or human kidney 293T cells,respectively. Detailed analysis of in vivo or in vitro folding of these mutant proteins revealed that one or two amino acid residues at the N terminus of pro-GCAP-II are critical, not only for the chaperone function in the folding but also for the net stabilization of pro-GCAP-II. In addition, size exclusion chromatography revealed that pro-GCAP-II exists as a dimer in solution. These data indicate that the propeptide has two roles in proper folding: the disulfide-coupled folding of the mature region and the dimerization of pro-GCAP-II.

Identification and recombinant production of human laminin α4 subunit splice variants

Laminins, the major basement membrane glycoproteins, are composed of three subunits. We identified a splice variant of the human laminin alpha4 subunit transcript containing 21 extra nucleotides. A heptapeptide sequence, MDCPTIS, was inserted close to the two cysteine residues possibly involved in the intersubunit disulfide bonds. Both the authentic alpha4 subunit (alpha4A) and the variant with the heptapeptide insertion (alpha4B) were readily secreted as laminin-8 trimers (alpha4Abeta1gamma1 or alpha4Bbeta1gamma1) upon cotransfection with expression vectors for the beta1 and gamma1 subunits. The purified recombinant laminin-8 containing the alpha4B subunit was more potent in promoting cell spreading than that containing alpha4A, raising the possibility that the alternative splicing of the alpha4 subunit transcript regulates the cell-adhesive activity of laminin-8. Since both alpha4A and alpha4B transcripts were detected by RT-PCR in several human cell lines, these two isoforms of laminin-8 with differing cell-adhesive activities are present in the basement membranes of human tissues.

GPR56 Functions Together with α3β1 Integrin in Regulating Cerebral Cortical Development

Loss of function mutations in GPR56, which encodes a G protein-coupled receptor, cause a specific human brain malformation called bilateral frontoparietal polymicrogyria (BFPP). Studies from BFPP postmortem brain tissue and Gpr56 knockout mice have previously showed that GPR56 deletion leads to breaches in the pial basement membrane (BM) and neuronal ectopias during cerebral cortical development. Since α3β1 integrin also plays a role in pial BM assembly and maintenance, we evaluated whether it functions together with GPR56 in regulating the same developmental process. We reveal that loss of α3 integrin enhances the cortical phenotype associated with Gpr56 deletion, and that neuronal overmigration through a breached pial BM occurs earlier in double knockout than in Gpr56 single knockout mice. These observations provide compelling evidence of the synergism of GPR56 and α3β1 integrin in regulating the development of cerebral cortex.

Nephronectin binds to heparan sulfate proteoglycans via its MAM domain.

Nephronectin is a basement membrane protein comprising five N-terminal epidermal growth factor (EGF)-like repeats, a central linker segment containing an Arg-Gly-Asp (RGD) motif and a C-terminal meprin-A5 protein-receptor protein tyrosine phosphatase μ (MAM) domain. Nephronectin has been shown to interact with α8β1 integrin through the central linker segment, but its interactions with other molecules remain to be elucidated. Here, we examined the binding of nephronectin to a panel of glycosaminoglycan (GAG) chains. Nephronectin bound strongly to heparin and chondroitin sulfate (CS)-E and moderately to heparan sulfate (HS), but failed to bind to CS-A, CS-C, CS-D, dermatan sulfate and hyaluronic acid. Deletion of the MAM domain severely impaired the binding of nephronectin to heparin but not CS-E, whereas deletion of the EGF-like repeats reduced its binding to CS-E but not heparin, suggesting that nephronectin interacts with CS-E and heparin through the EGF-like repeats and MAM domain, respectively. Consistent with these results, nephronectin bound to agrin and perlecan, which are heparan sulfate proteoglycans (HSPGs) in basement membranes, in HS-dependent manners. Site-directed mutagenesis of the MAM domain revealed that multiple basic amino acid residues in the putative loop regions were involved in the binding of the MAM domain to agrin. The binding of nephronectin to basement membrane HSPGs was further confirmed by in situ nephronectin overlay assays using mouse frozen tissue sections. Taken together, these findings indicate that nephronectin is capable of binding to HSPGs in basement membranes via the MAM domain, and thereby raise the possibility that interactions with basement membrane HSPGs may be involved in the deposition of nephronectin onto basement membranes.

Identification and characterization of nCLP2, a novel C1q family protein expressed in the central nervous system

The C1q family is characterized by the C-terminally conserved globular C1q (gC1q) domain. Although more than 30 C1q family proteins have been identified in mammals, many of them remain ill-defined with respect to their molecular and biological properties. Here, we report on a novel C1q family protein specifically expressed in the central nervous system (CNS), which we designated neural C1q-like protein (nCLP) 2. nCLP2 was secreted as disulphide-bonded multimers comprising trimeric units. The multimers were stabilized by interchain disulphide bonds involving the cysteine residues in the N-terminal variable region and the C-terminal gC1q domain. The expression of nCLP2 was restricted to several brain regions and retina, including regions associated with memory formation (i.e. hippocampus, entorhinal cortex, anterodorsal thalamic nucleus). Immunoelectron microscopy revealed that nCLP2 was localized in the mossy fibre axons of hippocampal granule cells and their synaptic boutons and clefts, implying that nCLP2 was anterogradely transported in mossy fibres and secreted from the presynaptic termini. These results suggest that nCLP2 plays roles in synaptic function and maintenance in the CNS.

Differential effects of laminin isoforms on axon and dendrite development in hippocampal neurons

Laminins play an important role in neuronal development. However, the effects of each laminin isoform on neurite morphology remain unclear. Here, we examined the effects of particular laminin (LN) isoforms on hippocampal neuron morphology. We found that LN-511 remarkably promoted elongation of both axons and dendrites, but reduced the number of dendrites. LN-511 E8 fragment, which includes the integrin-binding region, promoted axon and dendrite outgrowth and increased the dendrite number, although its effect on axon and dendrite elongation was smaller than that of full-length LN-511. These results suggest that LN-511 regulates axon and dendrite development by integrin-dependent and -independent mechanisms.

Polydom Is an Extracellular Matrix Protein Involved in Lymphatic Vessel Remodeling

Rationale: Lymphatic vasculature constitutes a second vascular system essential for immune surveillance and tissue fluid homeostasis. Maturation of the hierarchical vascular structure, with a highly branched network of capillaries and ducts, is crucial for its function. Environmental cues mediate the remodeling process, but the mechanism that underlies this process is largely unknown. Objective: Polydom (also called Svep1) is an extracellular matrix protein identified as a high-affinity ligand for integrin &agr;9&bgr;1. However, its physiological function is unclear. Here, we investigated the role of Polydom in lymphatic development. Methods and Results: We generated Polydom-deficient mice. Polydom−/− mice showed severe edema and died immediately after birth because of respiratory failure. We found that although a primitive lymphatic plexus was formed, it failed to undergo remodeling in Polydom−/− embryos, including sprouting of new capillaries and formation of collecting lymphatic vessels. Impaired lymphatic development was also observed after knockdown/knockout of polydom in zebrafish. Polydom was deposited around lymphatic vessels, but secreted from surrounding mesenchymal cells. Expression of Foxc2 (forkhead box protein c2), a transcription factor involved in lymphatic remodeling, was decreased in Polydom−/− mice. Polydom bound to the lymphangiogenic factor Ang-2 (angiopoietin-2), which was found to upregulate Foxc2 expression in cultured lymphatic endothelial cells. Expressions of Tie1/Tie2 receptors for angiopoietins were also decreased in Polydom−/− mice. Conclusions: Polydom affects remodeling of lymphatic vessels in both mouse and zebrafish. Polydom deposited around lymphatic vessels seems to ensure Foxc2 upregulation in lymphatic endothelial cells, possibly via the Ang-2 and Tie1/Tie2 receptor system.

The mechanism of the propeptide-mediated folding of guanylyl cyclase activating peptides

Guanylyl cyclase activating peptides (GCAPs), endogenous ligands for guanylyl cyclase C, are initially secreted in the form of prohormones and then processed into mature forms (Fig. 1). In previous studies, we have shown that the mature forms, GCAPs, do not contain the requisite information to permit themselves to adopt the native disulfide pairing, and that the pro-leader peptides in the precursor proteins mediate the folding of the mature peptides, resulting in the correct disulfide pairing in the mature peptides [1,2].