Integrins alpha1beta1 and alpha2beta1 are receptors for the rotavirus enterotoxin.

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 alpha1beta1 and alpha2beta1 are receptors for NSP4. NSP4 specifically binds to the alpha1 and alpha2 I domains with apparent K(d) = 1-2.7 muM. 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, E(120)A, fails to bind alpha2 integrin I domain. NSP4 has two distinct integrin interaction domains. NSP4 amino acids 114-130 are essential for binding to the I domain, and NSP4 peptide 114-135 blocks binding of the natural ligand, collagen I, to integrin alpha2. NSP4 amino acids 131-140 are not associated with the initial binding to the I domain, but elicit signaling that leads to the spreading of attached C2C12-alpha2 cells, mouse myoblast cells stably expressing the human alpha2 integrin. NSP4 colocalizes with integrin alpha2 on the basolateral surface of rotavirus-infected polarized intestinal epithelial (Caco-2) cells as well as surrounding noninfected cells. NSP4 mutants that fail to bind or signal through integrin alpha2 were attenuated in diarrhea induction in neonatal mice. These results indicate that NSP4 interaction with integrin alpha1 and alpha2 is an important component of enterotoxin function and rotavirus pathogenesis, further distinguishing this viral virulence factor from other microbial enterotoxins.

Scl1-dependent internalization of group A Streptococcus via direct interactions with the alpha2beta(1) integrin enhances pathogen survival and re-emergence.

The 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 alpha(2)beta(1). Here, we investigate whether the same Scl1 variant expressed by GAS cells interacts with the integrin alpha2beta(1) and affects the biological outcome of host-pathogen interactions. We demonstrate that GAS adherence and internalization involve direct interactions between surface expressed Scl1 and the alpha2beta(1) 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-alpha2 integrin-subunit antibody and type I collagen, (iii) recombinant alpha2-I domain bound the wild-type GAS cells and (iv) internalization of wild-type cells was significantly increased in C2C12 cells expressing the alpha2beta(1) 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.

Cryptogenic liver disease in four children: a novel congenital disorder of glycosylation.

We investigated the metabolic defect(s) of four children who presented with isolated cryptogenic chronic liver disease, coagulopathy, and abnormalities of several unrelated serum glycoproteins. Analysis of the patients' serum glycoproteins and fibroblasts suggest they have a novel congenital disorder of glycosylation (CDG). All had abnormal transferrin (Tf) isoelectric focusing (IEF) profiles. More detailed analysis of Tf by electrospray ionization mass spectrometry (ESI-MS) showed a plethora of abnormal glycosylations that included loss of 1-2 sialic acids and 1-2 galactose units, typical of Group II defects. Tf from two patients also lacked 1-2 entire oligosaccharide chains, typical of Group One disorders. Total serum N-glycans were analyzed by HPLC and matrix-assisted laser desorption/ionization mass spectrometry and also showed increased proportion of neutral glycan chains lacking sialic acids and galactose units. Analysis of patient fibroblasts eliminated CDG-Ia, through CDG-Ih, -IL and CDG-IId. Our results suggest that a subset of children with clinically asymptomatic, cryptogenic hypertransaminasemia and/or liver steato-fibrosis may represent a novel type of CDG-X with an unknown defect(s). Clinicians are encouraged to test such patients for abnormal Tf glycosylation by ESI-MS.

Decorin core protein secretion is regulated by N-linked oligosaccharide and glycosaminoglycan additions.

Expression of decorin using the vaccinia virus/T7 expression system resulted in secretion of two distinct glycoforms: a proteoglycan substituted with a single chondroitin sulfate chain and N-linked oligosaccharides and a core protein glycoform substituted with N-linked glycans but without a glycosaminoglycan chain. In this report, we have addressed two distinct questions. What is the rate-limiting step in glycosaminoglycan synthesis? Is glycosylation with either N-linked oligosaccharides or glycosaminoglycan required for secretion of decorin? N-terminal sequencing of the core protein glycoform, the addition of benzyl-beta-d-xyloside, and a UDP-xylose: core protein beta-d-xylosyltransferase activity assay show that xylosylation is a rate-limiting step in chondroitin sulfate biosynthesis. Decorin can be efficiently secreted with N-linked oligosaccharides alone or with a single chondroitin sulfate chain alone; however, there is severely impaired secretion of core protein devoid of any glycosylation. A decorin core protein mutant devoid of N-linked oligosaccharide attachment sites will not be secreted by Chinese hamster ovary cells deficient in xylosyltransferase or by parental Chinese hamster ovary wild type cells if the xylosyltransferase recognition sequence is disrupted. This finding suggests that quality control mechanisms sensitive to an absence of N-linked oligosaccharides can be abrogated by interaction of the core protein with the glycosaminoglycan synthetic machinery. We propose a model of regulation of decorin secretion that has several components, including appropriate substitution with N-linked oligosaccharides and factors involved in glycosaminoglycan synthesis.

Molecular and clinical description of the first US patients with congenital disorder of glycosylation Ig.

In this report we describe the first two US patients with congenital disorder of glycosylation type Ig (CDG-Ig). Both patients presented with symptoms indicating CDG, including developmental delay, hypotonia and failure to thrive, and tested positive for deficient glycosylation of transferrin. Labeling of the patients' lipid-linked oligosaccharides suggested mutations in the hALG12 gene, encoding a mannosyltransferase. Both patients were shown to carry previously unpublished hALG12-mutations. Patient 1 has one allele with a deletion of G29, resulting in a premature stop codon, and another allele with an 824G>A mutation yielding an S275N amino acid change. Patient 2 carries two heterozygous mutations (688T>G and 931C>T), resulting in two amino acid exchanges, Y230D and R311C. An adenoviral vector expressing wild type hALG12 corrects the abnormal lipid-linked oligosaccharide pattern of the patients' cells. In addition to common CDG symptoms, these patients also presented with low IgG and genital hypoplasia, symptoms previously described in CDG-Ig patients. We therefore conclude that a combination of developmental delay, low IgG, and genital hypoplasia should prompt CDG testing.

Bone morphogenetic protein-1/tolloid-related metalloproteinases process osteoglycin and enhance its ability to regulate collagen fibrillogenesis.

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-beta 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 pro- and 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.