Candidate loci shared among periodontal disease, diabetes and bone density

IntroductionWhile periodontal disease (PD) has been associated with type 2 diabetes (T2D) and osteoporosis, the underlying genetic mechanisms for these associations remain largely unknown. The aim of this study is to apply cross-trait genetic analyses to investigate the potentially shared biology among PD, T2D, and bone mineral density (BMD) by assessing pairwise genetic correlations and searching for shared polymorphisms.MethodsWe applied cross-trait genetic analyses leveraging genome-wide association study (GWAS) summary statistics for: Periodontitis/loose teeth from the UKBB/GLIDE consortium (PerioLT, N=506594), T2D from the DIAGRAM consortium (Neff=228825), and BMD from the GEFOS consortium (N=426824). Among all three, pair-wise genetic correlations were estimated with linkage disequilibrium (LD) score regression. Multi-trait meta-analysis of GWAS (MTAG) and colocalization analyses were performed to discover shared genome-wide significant variants (pMTAG <5x10-8). For replication, we conducted independent genetic analyses in the Women’s Genome Health Study (WGHS), a prospective cohort study of middle-aged women of whom 14711 provided self-reported periodontal disease diagnosis, oral health measures, and periodontal risk factor data including incident T2D.ResultsSignificant genetic correlations were identified between PerioLT/T2D (Rg=0.23; SE=0.04; p=7.4e-09) and T2D/BMD (Rg=0.09; SE=0.02; p=9.8e-06). Twenty-one independent pleiotropic variants were identified via MTAG (pMTAG<5x10-8 across all traits). Of these variants, genetic signals for PerioLT and T2D colocalized at one candidate variant (rs17522122; ProbH4 = 0.58), a 3’UTR variant of AKAP6. Colocalization between T2D/BMD and the original PerioLT GWAS p-values suggested 14 additional loci. In the independent WGHS sample, which includes responses to a validated oral health questionnaire for PD surveillance, the primary shared candidate (rs17522122) was associated with less frequent dental flossing [OR(95%CI)= 0.92 (0.87-0.98), p=0.007], a response that is correlated with worse PD status. Moreover, 4 additional candidate variants were indirectly supported by associations with less frequent dental flossing [rs75933965, 1.17(1.04-1.31), p=0.008], less frequent dental visits [rs77464186, 0.82(0.75-0.91), p=0.0002], less frequent dental prophylaxis [rs67111375, 0.91(0.83-0.99), p=0.03; rs77464186, 0.80(0.72-0.89), p=3.8e-05], or having bone loss around teeth [rs8047395, 1.09(1.03-1.15), p=0.005].DiscussionThis integrative approach identified one colocalized locus and 14 additional candidate loci that are shared between T2D and PD/oral health by comparing effects across PD, T2D and BMD. Future research is needed to independently validate our findings

Characterization of the human gelatinase : a collagen binding domain

Matrix raetalloproteinases (MMPs) can collectively degrade most extracellular matrix components during normal tissue remodeling and have also been implicated in pathological inflammatory diseases and in tumor cell invasive growth. Exploring the MMP-ligand interactions is important in understanding the function of these enzymes. Gelatinase A, like the other MMPs, is composed of distinct functional domains. One domain unique to the gelatinases consists of three fibronectin type II-like modules. The type II modules in fibronectin provide this molecule with gelatin binding properties. Therefore, the hypothesis was formulated that the fibronectin type II-like modules also provide gelatinase A with ligand binding. The tri-modular recombinant collagen binding domain of human gelatinase A (rCBD123) expressed in E. coli bound native type I collagen as well as denatured types I, IV and V collagen, elastin, and heparin. All of the gelatinase A type I collagen binding properties were found to reside in the CBD. However, rCBD123 did not bind several substrates including native type V collagen or fibronectin. Although gelatinase A can degrade basement membrane, rCBD123 did not bind laminin, fibronectin, SPARC, or matrigel. Binding site analysis further revealed that rCBD123 can bind at least two collagen molecules simultaneously. Whereas the major binding site in native type I collagen is in the telopeptide ends, collagen denaturation exposes multiple binding sites. Lysine residues were found to be important molecular determinants for ligand interactions of the CBD. Acetylation of rCBD123 lysine residues abolished heparin binding and reduced binding to collagen. Site-specific substitution of rCBD123 lysines with alanines demonstrated that K357 in the third module is required for heparin binding. Unaltered binding to other ligands by K357A and no change from wildtype protein in secondary structural components, as assessed by circular dichroism spectral analysis, confirmed that the loss of heparin binding was not a result of structural perturbation. These results together with structure modeling of the gelatinase A CBD indicated that two or more modules are required for heparin binding. Another mutant, K263A, demonstrated reduced saturation level binding to collagen but with an unchanged Kd for the interaction pointing to the presence of more than one collagen binding sites in the domain. Studies of mechanisms for gelatinase A cell surface localization showed specific cell binding to coated rCBD123 which was inhibited by preincubation of cells with soluble rCBD123. The cellular proteins binding rCBD123 were characteristic of collagen by electrophoretic behavior and resistance to digestion by pepsin but not bacterial collagenase. In addition, cell binding to rCBD123 was abolished by treatment of the cells with collagenase, and was reduced on a collagen binding deficient mutant of rCBD123. That rCBD123 could compete progelatinase A from cultured human gingival fibroblasts and that cell binding to rCBD123 was blocked by a Bl-integrin specific antibody point to the formation of a gelatinase A/ native type I collagen/ 61-integrin cell surface attachment complex. Thus, the CBD is an essential gelatinase A ligand binding domain. CBD lysine residues are important molecular determinants of substrate specificities and module cooperativity is likely required for the.ligand interactions. Gelatinase A can be positioned via the CBD to cell surfaces where it may be stored poised for activation and proteolysis.Dentistry, Faculty ofOral Biological and Medical Sciences (OBMS), Department ofGraduat

The involvement of the fibronectin type II-like modules of human gelatinase A in cell surface localization and activation

Recombinant collagen-binding domain (rCBD) comprising the three fibronectin type II-like modules of human gelatinase A was found to compete the zymogen form of this matrix metalloproteinase from the cell surface of normal human fibroblasts in culture. Upon concanavalin A treatment of cells, the induced cellular activation of gelatinase A was markedly elevated in the presence of the rCBD. Therefore, the mechanistic aspects of gelatinase A binding to cells by this domain were further studied using cell attachment assays. Fibroblasts attached to rCBD-coated microplate wells in a manner that was inhibited by soluble rCBD, blocking antibodies to the � 1-integrin subunit but not the � 2-integrin subunit, and bacterial collagenase treatment. Addition of soluble collagen rescued the attachment o

Intraspecific variation in aerobic and anaerobic locomotion: gilthead sea bream (Sparus aurata) and Trinidadian guppy (Poecilia reticulata) do not exhibit a trade-off between maximum sustained swimming speed and minimum cost of transport.

Intraspecific variation and trade-off in aerobic and anaerobic traits remain poorly understood in aquatic locomotion. Using gilthead sea bream (Sparus aurata) and Trinidadian guppy (Poecilia reticulata), both axial swimmers, this study tested four hypotheses: 1) gait transition from steady to unsteady (i.e. burst-assisted) swimming is associated with anaerobic metabolism evidenced as excess post exercise oxygen consumption (EPOC); 2) variation in swimming performance (critical swimming speed; Ucrit) correlates with metabolic scope (MS) or anaerobic capacity (i.e. maximum EPOC); 3) there is a trade-off between maximum sustained swimming speed (Usus) and minimum cost of transport (COTmin); and 4) variation in Usus correlates positively with optimum swimming speed (Uopt; i.e. the speed that minimizes energy expenditure per unit of distance travelled). Data collection involved swimming respirometry and video analysis. Results showed that anaerobic swimming costs (i.e. EPOC) increase linearly with the number of bursts in S. aurata, with each burst corresponding to 0.53 mg O2 kg-1. Data are consistent with a previous study on striped surfperch (Embiotoca lateralis), a labriform swimmer, suggesting that the metabolic cost of burst swimming is similar across various types of locomotion. There was no correlation between Ucrit and MS or anaerobic capacity in S. aurata indicating that other factors, including morphological or biomechanical traits, influenced Ucrit. We found no evidence of a trade-off between Usus and COTmin. In fact, data revealed significant negative correlations between Usus and COTmin, suggesting that individuals with high Usus also exhibit low COTmin. Finally, there were positive correlations between Usus and Uopt. Our study demonstrates the energetic importance of anaerobic metabolism during unsteady swimming, and provides intraspecific evidence that superior maximum sustained swimming speed is associated with superior swimming economy and optimum speed

Inhibition of MMP-2 gelatinolysis by targeting exodomain–substrate interactions

MMP-2 (matrix metalloproteinase 2) contains a CBD (collagen-binding domain), which is essential for positioning gelatin substrate molecules relative to the catalytic site for cleavage. Deletion of the CBD or disruption of CBD-mediated gelatin binding inhibits gelatinolysis by MMP-2. To identify CBD-binding sites on type I collagen and collagen peptides with the capacity to compete CBD binding of gelatin and thereby inhibit gelatinolysis by MMP-2, we screened a one-bead one-peptide combinatorial peptide library with recombinant CBD as bait. Analyses of sequences from the CBD-binding peptides pointed to residues 715–721 in human α1(I) collagen chain as a binding site for CBD. A peptide (P713) including this collagen segment was synthesized for analyses. In SPR (surface plasmon resonance) assays, the CBD and MMP-2E404A, a catalytically inactive MMP-2 mutant, both bound immobilized P713 in a concentration-dependent manner, but not a scrambled control peptide. Furthermore, P713 competed gelatin binding by the CBD and MMP-2E404A. In control assays, neither of the non-collagen binding alkylated CBD or MMP-2 with deletion of CBD (MMP-2ΔCBD) bound P713. Consistent with the exodomain functions of the CBD, P713 inhibited ∼90% of the MMP-2 gelatin cleavage, but less than 20% of the MMP-2 activity on a peptide substrate (NFF-1) which does not require the CBD for cleavage. Confirming the specificity of the inhibition, P713 did not alter MMP-2ΔCBD or MMP-8 activities. These experiments identified a CBD-binding site on type I collagen and demonstrated that a corresponding synthetic peptide can inhibit hydrolysis of type I and IV collagens by competing CBD-mediated gelatin binding to MMP-2

Functional basis for the overlap in ligand interactions and substrate specificities of matrix metalloproteinases-9 and -2

The MMPs (matrix metalloproteinases) MMP-9 and -2 each possess a unique CBD (collagen-binding domain) containing three fibronectin type II-like modules. The present experiments investigated whether the contributions to ligand interactions and enzymatic activities by the CBD of MMP-9 (CBD-9) corresponded to those of CBD in MMP-2 (CBD-2). The interactions of recombinant CBD-9 with a series of collagen types and extracellular matrix molecules were characterized by protein–protein binding assays. CBD-9 bound native and denatured type I, II, III, IV and V collagen, as well as Matrigel and laminin, with apparent K(d) values of (0.1–6.8)×10(−7) M, which were similar to the K(d) values for CBD-2 [(0.2–3.7)×10(−7) M]. However, CBD-9 bound neither native nor denatured type VI collagen. We also generated two modified MMPs, MMP-9(E402A) and MMP-2(E404A), by site-specific mutations in the active sites to obtain enzymes with intact ligand binding, but abrogated catalytic properties. In subsequent competitive binding assays, CBD-9 and MMP-9(E402A) inhibited the interactions of MMP-2(E404A) and, conversely, CBD-2 and MMP-2(E404A) competed with MMP-9(E402A) binding to native and denatured type I collagens, pointing to shared binding sites. Importantly, the capacity of CBD-9 to disrupt the MMP-9 and MMP-2 binding of collagen translated to inhibition of the gelatinolytic activity of the enzymes. Collectively, these results emphasize the essential contribution of CBD-9 to MMP-9 substrate binding and gelatinolysis, and demonstrate that the CBDs of MMP-9 and MMP-2 bind the same or closely positioned sites on type I collagen