Phenotypic characterization of epiphycan-deficient and epiphycan/biglycan double-deficient mice.

Objective: To characterize the in vivo role epiphycan (Epn) has in cartilage development and/or maintenance.

Methods: Epn-deficient mice were generated by disrupting the Epn gene in mouse embryonic stem cells. Epn/biglycan (Bgn) double-deficient mice were produced by crossing Epn-deficient mice with Bgn-deficient mice.

A family of fibrinogen-binding MSCRAMMs from Enterococcus faecalis.

We report that three (EF0089, EF2505 and EF1896, renamed here Fss1, Fss2 and Fss3, respectively, for Enterococcus faecalis surface protein) of the recently predicted MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) in E. faecalis strain V583 bind fibrinogen (Fg). Despite an absence of extensive primary sequence homology, the three proteins appear to be related structurally.

A structural model of the Staphylococcus aureus ClfA-fibrinogen interaction opens new avenues for the design of anti-staphylococcal therapeutics.

The fibrinogen (Fg) binding MSCRAMM Clumping factor A (ClfA) from Staphylococcus aureus interacts with the C-terminal region of the fibrinogen (Fg) gamma-chain. ClfA is the major virulence factor responsible for the observed clumping of S. aureus in blood plasma and has been implicated as a virulence factor in a mouse model of septic arthritis and in rabbit and rat models of infective endocarditis.

In vivo demonstration and quantification of intracellular Bacillus anthracis in lung epithelial cells.

Inhalational anthrax is initiated by the entry of Bacillus anthracis spores into the lung. A critical early event in the establishment of an infection is the dissemination of spores from the lung. Using in vitro cell culture assays, we previously demonstrated that B.

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.

Potential dissemination of Bacillus anthracis utilizing human lung epithelial cells.

Dissemination of Bacillus anthracis spores from the lung is a critical early event in the establishment of inhalational anthrax. We recently reported that B. anthracis could adhere to and be internalized by cultured intestinal epithelial and fibroblast cells.

Evidence for the "dock, lock, and latch" ligand binding mechanism of the staphylococcal microbial surface component recognizing adhesive matrix molecules (MSCRAMM) SdrG.

Staphylococcus epidermidis is an opportunistic pathogen and a major cause of foreign body infections. The S. epidermidis fibrinogen (Fg)-binding adhesin SdrG is necessary and sufficient for the attachment of this pathogen to Fg-coated materials.

Bacillus anthracis internalization by human fibroblasts and epithelial cells.

The current model for Bacillus anthracis dissemination in vivo focuses on macrophages as carriers. However, recent evidence suggested that other host cells may also play a role in the process. Here, we tested the possibility of B.

The Enterococcus faecalis MSCRAMM ACE binds its ligand by the Collagen Hug model.

We have determined the crystal structure of the ligand binding segment of the Enterococcus faecalis collagen binding MSCRAMM ACE (microbial surface components recognizing adhesive matrix molecules adhesin of collagen from enterococci). This segment is composed of two subdomains, N(1) and N(2), each adopting an IgG-like fold and forming a putative collagen binding surface at the interface between the two subdomains. This structure is very similar to that recently reported for CNA, the collagen binding MSCRAMM of Staphylococcus aureus, for which a unique ligand binding mechanism called the Collagen Hug was proposed.

Staphylococcus aureus Panton-Valentine leukocidin causes necrotizing pneumonia.

The Staphylococcus aureus Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by strains epidemiologically associated with the current outbreak of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) and with the often-lethal necrotizing pneumonia. To investigate the role of PVL in pulmonary disease, we tested the pathogenicity of clinical isolates, isogenic PVL-negative and PVL-positive S. aureus strains, as well as purified PVL, in a mouse acute pneumonia model.

Decorin modulates fibrin assembly and structure.

Emerging evidence indicates that fibrin clotting is regulated by different external factors. We demonstrated recently that decorin, a regulator of collagen fibrillogenesis and transforming growth factor-beta activity, binds to the D regions of fibrinogen (Dugan, T.A.

Assessment of prokaryotic collagen-like sequences derived from streptococcal Scl1 and Scl2 proteins as a source of recombinant GXY polymers.

Collagen triple helix, composed of the repeating Gly-Xaa-Yaa (GXY) sequence, is a structural element found in all multicellular animals and also in some prokaryotes. Long GXY polymers are highly regarded components used in food, cosmetic, biomedical, and pharmaceutical industries. In this study, we explore a new concept for the production of recombinant GXY polymers which are based on the sequence of "prokaryotic collagens", the streptococcal collagen-like proteins Scl1 and Scl2.

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.

Murine lipid phosphate phosphohydrolase-3 acts as a cell-associated integrin ligand.

Lipid phosphate phosphohydrolase-3 (LPP3) is a cell surface protein that exhibits ectoenzyme activity. Previously, we identified human LPP3 in a functional assay of angiogenesis and showed that the Arg-Gly-Asp (RGD) motif in the proposed second extracellular domain interacts with a subset of integrins to mediate cell-cell adhesion. In contrast to the RGD domain of human LPP3, murine Lpp3 contains a variant sequence, Arg-Gly-Glu (RGE).

A novel binding site in collagen type III for integrins alpha1beta1 and alpha2beta1.

Previously identified high affinity integrin-binding motifs in collagens, GFOGER and GLOGER, are not present in type III collagen. Here, we first characterized the binding of recombinant I domains from integrins alpha(1) and alpha(2) (alpha(1)I and alpha(2)I) to fibrillar collagen types I-III and showed that each I domain bound to the three types of collagens with similar affinities. Using rotary shadowing followed by electron microscopy, we identified a high affinity binding region in human type III collagen recognized by alpha(1)I and alpha(2)I.

Multicomponent Lyme vaccine: three is not a crowd.

Lyme disease is caused by the spirochete Borrelia burgdorferi and it is the most common vector-borne disease in the United States. Disseminated spirochetes can persist in various tissues and can result in a variety of different disease manifestations. Vaccination trials testing various lipoprotein candidates have yielded mixed results despite the generation of robust antibody titers.

Identification and preliminary characterization of cell-wall-anchored proteins of Staphylococcus epidermidis.

Staphylococcus epidermidis is a ubiquitous human skin commensal that has emerged as a major cause of foreign-body infections. Eleven genes encoding putative cell-wall-anchored proteins were identified by computer analysis of the publicly available S. epidermidis unfinished genomic sequence.

Natural antisense mRNAs to hyaluronan synthase 2 inhibit hyaluronan biosynthesis and cell proliferation.

We report the identification of a natural antisense mRNA of hyaluronan synthase 2 that we have chosen to designate as HASNT (for HA synthase 2 antisense) in human and mouse. HASNT is transcribed from the opposite strand of the HAS2 gene locus and is represented by several independent expressed sequence tags in human. Portions of the mouse Hasnt gene were identified through an exon-trapping approach.

Recognizing scientific excellence in the biology of cell adhesion.

The prestigious 2005 Japan Prize for Cell Biology has been awarded to Dr. Masatoshi Takeichi, Director of RIKEN Developmental Biology, Kobe, Japan, and Dr. Erkki Ruoslahti, Distinguished Professor, The Burnham Institute, La Jolla, USA for their "Fundamental contribution in elucidating the molecular mechanisms of cell adhesion".

A streptococcal collagen-like protein interacts with the alpha2beta1 integrin and induces intracellular signaling.

The streptococcal collagen-like proteins Scl1 and Scl2 are prokaryotic members of a large protein family with domains containing the repeating amino acid sequence (Gly-Xaa-Yaa)(n) that form a collagen-like triple-helical structure. Here, we test the hypothesis that Scl variant might interact with mammalian collagen-binding integrins. We show that the recombinant Scl protein p176 promotes adhesion and spreading of human lung fibroblast cells through an alpha2beta1 integrin-mediated interaction as shown in cell adhesion inhibition assays using anti-alpha2beta1 and anti-beta1 integrins monoclonal antibodies.

Identification and biochemical characterization of two novel collagen binding MSCRAMMs of Bacillus anthracis.

Cell wall-anchored proteins play critical roles in the pathogenesis of infections caused by Gram-positive bacteria. Through the analysis of the genome of Bacillus anthracis Ames strain, we identified two novel putative cell wall-anchored proteins, BA0871 and BA5258, which have sequence homology to CNA, a cell wall-anchored collagen adhesin of Staphylococcus aureus. The two proteins have similar domain organization to that of CNA, with typical signal peptide sequences, a non-repetitive A region followed by repeats, and a characteristic cell wall-anchoring region.

Identification and characterization of the C3 binding domain of the Staphylococcus aureus extracellular fibrinogen-binding protein (Efb).

The secreted Staphylococcus aureus extracellular fibrinogen-binding protein (Efb) is a virulence factor that binds to both the complement component C3b and fibrinogen. Our laboratory previously reported that by binding to C3b, Efb inhibited complement activation and blocked opsonophagocytosis. We have now located the Efb binding domain in C3b to the C3d fragment and determined a disassociation constant (Kd) of 0.

BBK32, a fibronectin binding MSCRAMM from Borrelia burgdorferi, contains a disordered region that undergoes a conformational change on ligand binding.

BBK32 is a fibronectin-binding lipoprotein on Borrelia burgdorferi, the causative agent of Lyme disease. Analysis using secondary structure prediction programs suggested that BBK32 is composed of two domains, an N-terminal segment lacking well defined secondary structure and a C-terminal segment composed largely of alpha-helices. Analysis of purified recombinant forms of the two domains by circular dichroism spectroscopy, gel permeation chromatography, and intrinsic viscosity determination were consistent with an N-terminal-extended, unstructured segment and a C-terminal globular domain in BBK32.