RESUMO
Bone biomineralization is a complex process in which type I collagen and associated non-collagenous proteins (NCPs), including glycoproteins and proteoglycans, interact closely with inorganic calcium and phosphate ions to control the precipitation of nanosized, non-stoichiometric hydroxyapatite (HAP, idealized stoichiometry Ca10(PO4)6(OH)2) within the organic matrix of a tissue. The ability of certain vertebrate tissues to mineralize is critically related to several aspects of their function. The goal of this study was to identify specific NCPs in mineralizing and non-mineralizing tissues of two animal models, rat and turkey, and to determine whether some NCPs are unique to each type of tissue. The tissues investigated were rat femur (mineralizing) and tail tendon (non-mineralizing) and turkey leg tendon (having both mineralizing and non-mineralizing regions in the same individual specimen). An experimental approach ex vivo was designed for this investigation by combining sequential protein extraction with comprehensive protein mapping using proteomics and Western blotting. The extraction method enabled separation of various NCPs based on their association with either the extracellular organic collagenous matrix phases or the inorganic mineral phases of the tissues. The proteomics work generated a complete picture of NCPs in different tissues and animal species. Subsequently, Western blotting provided validation for some of the proteomics findings. The survey then yielded generalized results relevant to various protein families, rather than only individual NCPs. This study focused primarily on the NCPs belonging to the small leucine-rich proteoglycan (SLRP) family and the small integrin-binding ligand N-linked glycoproteins (SIBLINGs). SLRPs were found to be associated only with the collagenous matrix, a result suggesting that they are mainly involved in structural matrix organization and not in mineralization. SIBLINGs as well as matrix Gla (γ-carboxyglutamate) protein were strictly localized within the inorganic mineral phase of mineralizing tissues, a finding suggesting that their roles are limited to mineralization. The results from this study indicated that osteocalcin was closely involved in mineralization but did not preclude possible additional roles as a hormone. This report provides for the first time a spatial survey and comparison of NCPs from mineralizing and non-mineralizing tissues ex vivo and defines the proteome of turkey leg tendons as a model for vertebrate mineralization.
RESUMO
Streptococcus pyogenes is a major human pathogen that causes a variety of diseases ranging from mild skin and throat infections to fatal septicemia. In severe invasive infections, S. pyogenes encounters and interacts with components of the extracellular matrix (ECM), including small leucine rich-proteoglycans (SLRPs). In this study, we report a novel antimicrobial role played by SLRPs biglycan, decorin, fibromodulin and osteoadherin, specifically in promoting the eradication of S. pyogenes in a human sepsis model of infection. SLRPs can be released from the ECM and de novo synthesized by a number of cell types. We reveal that infection of human monocytes by S. pyogenes induces the expression of decorin. Furthermore, we show that the majority of genetically distinct and clinically relevant S. pyogenes isolates interact with SLRPs resulting in decreased survival in blood killing assays. Biglycan and decorin induce TLR2 and TLR4 signaling cascades resulting in secretion of proinflammatory and chemotactic molecules and recruitment of professional phagocytes. Surprisingly, SLRP-mediated elimination of S. pyogenes occurs independently of TLR activation. Our results indicate that SLRPs act in concert with human serum, enhancing deposition of complement activation fragments and the classical activator C1q on the bacterial surface, facilitating efficient microbial eradication. Addition of the complement C3 inhibitor compstatin significantly reverses SLRP-induced blood killing, confirming active complement as a key mediator in SLRP-mediated bacterial destruction. Taken together our results add to the functional repertoire of SLRPs, expanding to encompass their role in controlling bacterial infection.