SummaryThe molecular pathogenesis of infections caused by group A Streptococcus (GAS) is not fully understood. We recently reported that a recombinant protein derived from the collagen-like surface protein, Scl1, bound to the human collagen receptor, integrin a2b1. Here, we investigate whether the same Scl1 variant expressed by GAS cells interacts with the integrin a2b1 and affects the biological outcome of hostpathogen interactions. We demonstrate that GAS adherence and internalization involve direct interactions between surface expressed Scl1 and the a2b1 integrin, because (i) both adherence and internalization of the scl1-inactivated mutant were significantly decreased, and were restored by in-trans complementation of Scl1 expression, (ii) GAS internalization was reduced by pre-treatment of HEp-2 cells with anti-a2 integrin-subunit antibody and type I collagen, (iii) recombinant a2-I domain bound the wild-type GAS cells and (iv) internalization of wild-type cells was significantly increased in C2C12 cells expressing the a2b1 integrin as the only collagen-binding integrin. Next, we determined that internalized GAS re-emerges from epithelial cells into the extracellular environment. Taken together, our data describe a new molecular mechanism used by GAS involving the direct interaction between Scl1 and integrins, which increases the overall capability of the pathogen to survive and re-emerge.

The mammalian bone morphogenetic protein-1 (BMP-1)/Tolloid-related metalloproteinases play key roles in regulating formation of the extracellular matrix (ECM) via biosynthetic processing of various precursor proteins into mature functional enzymes, structural proteins, and proteins involved in initiating the mineralization of hard tissue ECMs. They also have been shown to activate several members of the transforming growth factor-␤ superfamily, and may serve to coordinate such activation with formation of the ECM in morphogenetic events. Osteoglycin (OGN), a small leucine-rich proteoglycan with unclear functions, is found in cornea, bone, and other tissues, and appears to undergo proteolytic processing in vivo. Here we have successfully generated recombinant OGN and have employed it to demonstrate that a pro-form of OGN is processed to varying extents by all four mammalian BMP-1/Tolloid-like proteinases, to generate a 27-kDa species that corresponds to the major form of OGN found in cornea. Moreover, whereas wild-type mouse embryo fibroblasts (MEFs) produce primarily the processed, mature form of OGN, MEFs homozygous null for genes encoding three of the four mammalian BMP-1/Tolloid-related proteinases produce only unprocessed pro-OGN. Thus, all detectable pro-OGN processing activity in MEFs is accounted for by products of these genes. We also demonstrate that both proand mature OGN can regulate type I collagen fibrillogenesis, and that processing of the prodomain by BMP-1 potentiates the ability of OGN to modulate the formation of collagen fibrils.Bone morphogenetic protein-1 (BMP-1) 1 is the prototype of a class of structurally similar metalloproteinases that play various morphogenetic roles in a broad spectrum of species (1, 2).There are four mammalian members of this family: BMP-1, mammalian Tolloid (mTLD), and mammalian Tolloid-like 1 and 2 (mTLL-1 and mTLL-2) (3-5). BMP-1 and mTLD are encoded by alternatively spliced mRNAs of the same gene (3), whereas mTLL-1 and -2 are genetically distinct (4, 5). These proteinases play key roles in regulating formation of mammalian extracellular matrix (ECM), via biosynthetic processing of precursor proteins to form mature, functional matrix components. In the case of collagen fibers, this includes processing of the C-propeptides of procollagens I-III to yield the major fibrous components of ECM (5-9); proteolytic activation of the enzyme lysyl oxidase (10), which is necessary to the formation of covalent cross-links in collagen and elastic fibers (11); and processing of NH 2 -terminal globular domains, or in some cases C-propeptides, of minor fibrillar procollagen V and XI chains (12-14) to yield type V and XI monomers. Such monomers are incorporated into collagen types I and II fibrils, respectively, and appear to control the shapes and diameters of the resultant heterotypic collagen fibrils (15-18). Members of the same small group of proteinases also process precursors for laminin 5 (19, 20) and type VII collagen (21), both of which are involved in securing epithelia...

Matricellular proteins such as SPARC, thrombospondin 1 and 2, and tenascin C and X subserve important functions in extracellular matrix synthesis and cellular adhesion to extracellular matrix. By virtue of its reported interaction with collagen I and deadhesive activity on cells, we hypothesized that hevin, a member of the SPARC gene family, regulates dermal extracellular matrix and collagen fibril formation. We present evidence for an altered collagen matrix and levels of the proteoglycan decorin in the normal dermis and dermal wound bed of hevin-null mice. The dermal elastic modulus was also enhanced in hevin-null animals. The levels of decorin protein secreted by hevin-null dermal fibroblasts were increased by exogenous hevin in vitro, data indicating that hevin might regulate both decorin and collagen fibrillogenesis. We also report a decorin-independent function for hevin in collagen fibrillogenesis. In vitro fibrillogenesis assays indicated that hevin enhanced fibril formation kinetics. Furthermore, cell adhesion assays indicated that cells adhered differently to collagen fibrils formed in the presence of hevin. Our observations support the capacity of hevin to modulate the structure of dermal extracellular matrix, specifically by its regulation of decorin levels and collagen fibril assembly.

Rotavirus NSP4 is a viral enterotoxin capable of causing diarrhea in neonatal mice. This process is initiated by the binding of extracellular NSP4 to target molecule(s) on the cell surface that triggers a signaling cascade leading to diarrhea. We now report that the integrins ␣1␤1 and ␣2␤1 are receptors for NSP4. NSP4 specifically binds to the ␣1 and ␣2 I domains with apparent Kd ‫؍‬ 1-2.7 M. Binding is mediated by the I domain metal ion-dependent adhesion site motif, requires Mg 2؉ or Mn 2؉ , is abolished with EDTA, and an NSP4 point mutant, E120A, fails to bind ␣2 integrin I domain. NSP4 has two distinct integrin interaction domains. I domain ͉ diarrhea ͉ signaling ͉ NSP4 R otaviruses (RVs) are nonenveloped, triple-layered icosahedral viruses containing a genome of 11 segments of dsRNA that code for six structural (VP1, VP2, VP3, VP4, VP6, and VP7) and six nonstructural (NSP1-NSP6) proteins (1). RV is the leading etiologic agent of severe gastroenteritis in infants, young children, and animals worldwide. There is no known treatment to prevent RV transmission. Although new vaccines have recently shown promising results in clinical trials and are now licensed (2), vaccine efficacy in developing countries where disease prevention is needed most remains unknown. The recent recognition that RV causes extraintestinal infection in animals and children (3-6) highlights a continuing need to better understand the mechanisms of RV replication and pathogenesis. Increasing knowledge of the structure of RVs and protein function has supported new ideas about virus pathogenesis, virus assembly, and virus-host interactions (1,7,8). However, the molecular details of RV effects on intestinal epithelial cells or extraintestinal cells remain incompletely understood. NSP4 is the first described viral enterotoxin (9-11). The i.p. delivery of purified recombinant NSP4 or synthetic NSP4 peptides from mammalian and avian RVs that lack sequence similarity causes age-dependent diarrhea in young mice (9,(12)(13)(14)(15)(16). The cytoplasmic toxic domain of NSP4 (aa 112-175) is released from infected cells and hypothesized to interact with receptors on neighboring cells (9, 10, 17), mobilizing Ca 2ϩ by stimulating phospholipase C-mediated inositol 1,4,5-trisphosphate production (17), and eliciting age-dependent diarrhea in neonatal mice (9,18).Integrins are a family of heterodimeric cell surface receptors composed of ␣ and ␤ subunits involved in cell-cell, cellextracellular matrix, and cell-pathogen interactions (19)(20)(21)(22)(23)(24)(25). Vertebrates have nine ␣ subunits that contain a so-called I or A domain, in which a set of ␣ subunits (␣1, ␣2, ␣10, and ␣11) act as collagen receptors (21). The I domain forms a subset of the larger group of von Willebrand factor A (VWA) domains found in a wide range of proteins (26), and many appear to be involved in protein-protein interactions. The x-ray structures of recombinant ␣1, ␣2, ␣M, and ␣L I domains have defined a divalent cation-binding motif [metal ion-dependent adhesion site (MIDAS)] tha...

The dystrophin-associated protein complex (DAPC) links the cytoskeleton to the extracellular matrix, is essential for muscle cell survival, and is defective in a wide range of muscular dystrophies. The DAPC contains two transmembrane subcomplexes -the dystroglycans and the sarcoglycans. Although several extracellular binding partners have been identified for the dystroglycans, none have been described for the sarcoglycan subcomplex. Here we show that the small leucine-rich repeat proteoglycan biglycan binds to α-and γ-sarcoglycan as judged by ligand blot overlay and coimmunoprecipitation assays. Studies with biglycan-decorin chimeras show that α-and γ-sarcoglycan bind to distinct sites on the polypeptide core of biglycan. Both biglycan proteoglycan as well as biglycan polypeptide lacking glycosaminoglycan side chains are components of the dystrophin glycoprotein complex isolated from adult skeletal muscle membranes. Finally, immunohistochemical and biochemical studies with biglycan null mice show that the expression of α-and γ-sarcoglycan is selectively reduced in muscle from young (P14-P21) animals, while levels in adult muscle (≥P35) are unchanged. We conclude that biglycan is ligand for two members of the sarcoglycan complex and regulates their expression at discrete developmental ages.