Genomic editing of metformin efficacy-associated genetic variants in SLC47A1 does not alter SLC47A1 expression.

Several pharmacogenetics studies have identified an association between a greater metformin-dependent reduction in HbA1c levels and the minor A allele at rs2289669 in intron 10 of SLC47A1, encoding multidrug and toxin extrusion 1 (MATE1), a presumed metformin transporter. It is currently unknown if the rs2289669 locus is a cis-eQTL, which would validate its role as predictor of metformin efficacy. We looked at association between common genetic variants in the SLC47A1 gene region and HbA1c reduction after metformin treatment using locus-wise meta-analysis from the MetGen consortium. CRISPR-Cas9 was applied to perform allele editing of, or genomic deletion around, rs2289669 and of the closely linked rs8065082 in HepG2 cells. The genome-edited cells were evaluated for SLC47A1 expression and splicing. None of the common variants including rs2289669 showed significant association with metformin response. Genomic editing of either rs2289669 or rs8065082 did not alter SLC47A1 expression or splicing. Experimental and in silico analyses show that the rs2289669-containing haploblock does not appear to carry genetic variants that could explain its previously reported association with metformin efficacy.

Engineered allele substitution at PPARGC1A rs8192678 alters human white adipocyte differentiation, lipogenesis, and PGC-1α content and turnover.

AIMS/HYPOTHESIS: PPARGC1A encodes peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α), a central regulator of energy metabolism and mitochondrial function. A common polymorphism in PPARGC1A (rs8192678, C/T, Gly482Ser) has been associated with obesity and related metabolic disorders, but no published functional studies have investigated direct allele-specific effects in adipocyte biology. We examined whether rs8192678 is a causal variant and reveal its biological function in human white adipose cells. METHODS: We used CRISPR-Cas9 genome editing to perform an allelic switch (C-to-T or T-to-C) at rs8192678 in an isogenic human pre-adipocyte white adipose tissue (hWAs) cell line. Allele-edited single-cell clones were expanded and screened to obtain homozygous T/T (Ser482Ser), C/C (Gly482Gly) and heterozygous C/T (Gly482Ser) isogenic cell populations, followed by functional studies of the allele-dependent effects on white adipocyte differentiation and mitochondrial function. RESULTS: After differentiation, the C/C adipocytes were visibly less BODIPY-positive than T/T and C/T adipocytes, and had significantly lower triacylglycerol content. The C allele presented a dose-dependent lowering effect on lipogenesis, as well as lower expression of genes critical for adipogenesis, lipid catabolism, lipogenesis and lipolysis. Moreover, C/C adipocytes had decreased oxygen consumption rate (OCR) at basal and maximal respiration, and lower ATP-linked OCR. We determined that these effects were a consequence of a C-allele-driven dysregulation of PGC-1α protein content, turnover rate and transcriptional coactivator activity. CONCLUSIONS/INTERPRETATION: Our data show allele-specific causal effects of the rs8192678 variant on adipogenic differentiation. The C allele confers lower levels of PPARGC1A mRNA and PGC-1α protein, as well as disrupted dynamics of PGC-1α turnover and activity, with downstream effects on cellular differentiation and mitochondrial function. Our study provides the first experimentally deduced insights on the effects of rs8192678 on adipocyte phenotype.

The endoplasmic reticulum-resident collagen chaperone Hsp47 interacts with and promotes the secretion of decorin, fibromodulin, and lumican.

The build-up of diversified and tissue-specific assemblies of extracellular matrix (ECM) proteins depends on secreted and cell surface-located molecular arrays that coordinate ECM proteins into discrete designs. The family of small leucine-rich proteins (SLRPs) associates with and dictates the structure of fibrillar collagens, which form the backbone of most ECM types. However, whether SLRPs form complexes with proteins other than collagens is unclear. Here, we demonstrate that heat shock protein 47 (Hsp47), a well-established endoplasmic reticulum-resident collagen chaperone, also binds the SLRPs decorin, lumican, and fibromodulin with affinities comparable with that in the Hsp47-type I collagen interaction. Furthermore, we show that a lack of Hsp47 inhibits the cellular secretion of decorin and lumican. Our results expand the understanding of the concerted molecular interactions that control the secretion and organization of a functional collagenous ECM.

Inhibition of integrin αVß6 changes fibril thickness of stromal collagen in experimental carcinomas.

BACKGROUND: Chemotherapeutic efficacy can be improved by targeting the structure and function of the extracellular matrix (ECM) in the carcinomal stroma. This can be accomplished by e.g. inhibiting TGF-ß1 and -ß3 or treating with Imatinib, which results in scarcer collagen fibril structure in xenografted human KAT-4/HT29 (KAT-4) colon adenocarcinoma. METHODS: The potential role of αVß6 integrin-mediated activation of latent TGF-ß was studied in cultured KAT-4 and Capan-2 human ductal pancreatic carcinoma cells as well as in xenograft carcinoma generated by these cells. The monoclonal αVß6 integrin-specific monoclonal antibody 3G9 was used to inhibit the αVß6 integrin activity. RESULTS: Both KAT-4 and Capan-2 cells expressed the αVß6 integrin but only KAT-4 cells could utilize this integrin to activate latent TGF-ß in vitro. Only when Capan-2 cells were co-cultured with human F99 fibroblasts was the integrin activation mechanism triggered, suggesting a more complex, fibroblast-dependent, activation pathway. In nude mice, a 10-day treatment with 3G9 reduced collagen fibril thickness and interstitial fluid pressure in KAT-4 but not in the more desmoplastic Capan-2 tumors that, to achieve a similar effect, required a prolonged 3G9 treatment. In contrast, a 10-day direct inhibition of TGF-ß1 and -ß3 reduced collagen fibril thickness in both tumor models. CONCLUSION: Our data demonstrate that the αVß6-directed activation of latent TGF-ß plays a pivotal role in modulating the stromal collagen network in carcinoma, but that the sensitivity to αVß6 inhibition depends on the simultaneous presence of alternative paths for latent TGF-ß activation and the extent of desmoplasia.

The Tyrosine Sulfate Domain of Fibromodulin Binds Collagen and Enhances Fibril Formation.

Small leucine-rich proteoglycans interact with other extracellular matrix proteins and are important regulators of matrix assembly. Fibromodulin has a key role in connective tissues, binding collagen through two identified binding sites in its leucine-rich repeat domain and regulating collagen fibril formation in vitro and in vivo Some nine tyrosine residues in the fibromodulin N-terminal domain are O-sulfated, a posttranslational modification often involved in protein interactions. The N-terminal domain mimics heparin, binding proteins with clustered basic amino acid residues. Because heparin affects collagen fibril formation, we investigated whether tyrosine sulfate is involved in fibromodulin interactions with collagen. Using full-length fibromodulin and its N-terminal tyrosine-sulfated domain purified from tissue, as well as recombinant fibromodulin fragments, we found that the N-terminal domain binds collagen. The tyrosine-sulfated domain and the leucine-rich repeat domain both bound to three specific sites along the collagen type I molecule, at the N terminus and at 100 and 220 nm from the N terminus. The N-terminal domain shortened the collagen fibril formation lag phase and tyrosine sulfation was required for this effect. The isolated leucine-rich repeat domain inhibited the fibril formation rate, and full-length fibromodulin showed a combination of these effects. The fibrils formed in the presence of fibromodulin or its fragments showed more organized structure. Fibromodulin and its tyrosine sulfate domain remained bound on the formed fiber. Taken together, this suggests a novel, regulatory function for tyrosine sulfation in collagen interaction and control of fibril formation.

Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase.

The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.

The novel small leucine-rich protein chondroadherin-like (CHADL) is expressed in cartilage and modulates chondrocyte differentiation.

The constitution and biophysical properties of extracellular matrices can dramatically influence cellular phenotype during development, homeostasis, or pathogenesis. These effects can be signaled through a differentially regulated assembly of collagen fibrils, orchestrated by a family of collagen-associated small leucine-rich proteins (SLRPs). In this report, we describe the tissue-specific expression and function of a previously uncharacterized SLRP, chondroadherin-like (CHADL). We developed antibodies against CHADL and, by immunohistochemistry, detected CHADL expression mainly in skeletal tissues, particularly in fetal cartilage and in the pericellular space of adult chondrocytes. In situ hybridizations and immunoblots on tissue lysates confirmed this tissue-specific expression pattern. Recombinant CHADL bound collagen in cell culture and inhibited in vitro collagen fibrillogenesis. After Chadl shRNA knockdown, chondrogenic ATDC5 cells increased their differentiation, indicated by increased transcript levels of Sox9, Ihh, Col2a1, and Col10a1. The knockdown increased collagen II and aggrecan deposition in the cell layers. Microarray analysis of the knockdown samples suggested collagen receptor-related changes, although other upstream effects could not be excluded. Together, our data indicate that the novel SLRP CHADL is expressed in cartilaginous tissues, influences collagen fibrillogenesis, and modulates chondrocyte differentiation. CHADL appears to have a negative regulatory role, possibly ensuring the formation of a stable extracellular matrix.

Increased C-telopeptide cross-linking of tendon type I collagen in fibromodulin-deficient mice.

The controlled assembly of collagen monomers into fibrils, with accompanying intermolecular cross-linking by lysyl oxidase-mediated bonds, is vital to the structural and mechanical integrity of connective tissues. This process is influenced by collagen-associated proteins, including small leucine-rich proteins (SLRPs), but the regulatory mechanisms are not well understood. Deficiency in fibromodulin, an SLRP, causes abnormal collagen fibril ultrastructure and decreased mechanical strength in mouse tendons. In this study, fibromodulin deficiency rendered tendon collagen more resistant to nonproteolytic extraction. The collagen had an increased and altered cross-linking pattern at an early stage of fibril formation. Collagen extracts contained a higher proportion of stably cross-linked α1(I) chains as a result of their C-telopeptide lysines being more completely oxidized to aldehydes. The findings suggest that fibromodulin selectively affects the extent and pattern of lysyl oxidase-mediated collagen cross-linking by sterically hindering access of the enzyme to telopeptides, presumably through binding to the collagen. Such activity implies a broader role for SLRP family members in regulating collagen cross-linking placement and quantity.

Recurrent dominant mutations affecting two adjacent residues in the motor domain of the monomeric kinesin KIF22 result in skeletal dysplasia and joint laxity.

Spondyloepimetaphyseal dysplasia with joint laxity, leptodactylic type (lepto-SEMDJL, aka SEMDJL, Hall type), is an autosomal dominant skeletal disorder that, in spite of being relatively common among skeletal dysplasias, has eluded molecular elucidation so far. We used whole-exome sequencing of five unrelated individuals with lepto-SEMDJL to identify mutations in KIF22 as the cause of this skeletal condition. Missense mutations affecting one of two adjacent amino acids in the motor domain of KIF22 were present in 20 familial cases from eight families and in 12 other sporadic cases. The skeletal and connective tissue phenotype produced by these specific mutations point to functions of KIF22 beyond those previously ascribed functions involving chromosome segregation. Although we have found Kif22 to be strongly upregulated at the growth plate, the precise pathogenetic mechanisms remain to be elucidated.

Fibromodulin deficiency reduces low-density lipoprotein accumulation in atherosclerotic plaques in apolipoprotein E-null mice.

OBJECTIVE: The aim of this study was to analyze how an altered collagen structure affects development of atherosclerotic plaques. METHODS AND RESULTS: Fibromodulin-null mice develop an abnormal collagen fibril structure. In apolipoprotein E (ApoE)-null and ApoE/fibromodulin-null mice, a shear stress-modifying carotid artery cast induced formation of atherosclerotic plaques of different phenotypes; inflammatory in low-shear stress regions and fibrous in oscillatory shear stress regions. Electron microscopy showed that collagen fibrils were thicker and more heterogeneous in oscillatory shear stress lesions from ApoE/fibromodulin-null mice. Low-shear stress lesions were smaller in ApoE/fibromodulin-null mice and contained less lipids. Total plaque burden in aortas stained en face with Oil Red O, as well as lipid accumulation in aortic root lesions, was also decreased in ApoE/fibromodulin-null mice. In addition, lipid accumulation in RAW264.7 macrophages cultured on fibromodulin-deficient extracellular matrix was decreased, whereas levels of interleukin-6 and -10 were increased. Our results show that an abnormal plaque collagen fibril structure can influence atherosclerotic plaque development. CONCLUSIONS: The present findings suggest a more complex role for collagen in plaque stability than previously anticipated, in that it may promote lipid-accumulation and inflammation at the same time as it provides mechanical stability.

Human Genetic Variation at rs10071329 Correlates With Adiposity-Related Traits, Modulates PPARGC1B Expression, and Alters Brown Adipocyte Function.

Human genetic variation in PPARGC1B has been associated with adiposity, but the genetic variants that affect PPARGC1B expression have not been experimentally determined. Here, guided by previous observational data, we used clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) to scarlessly edit the alleles of the candidate causal genetic variant rs10071329 in a human brown adipocyte cell line. Switching the rs10071329 genotype from A/A to G/G enhanced PPARGC1B expression throughout the adipogenic differentiation, identifying rs10071329 as a cis-expression quantitative trait loci (eQTL). The higher PPARGC1B expression in G/G cells coincided with greater accumulation of triglycerides and higher expression of mitochondria-encoded genes, but without significant effects on adipogenic marker expression. Furthermore, G/G cells had improved basal- and norepinephrine-stimulated mitochondrial respiration, possibly relating to enhanced mitochondrial gene expression. The G/G cells also exhibited increased norepinephrine-stimulated glycerol release, indicating improved lipolysis. Altogether, our results showed that rs10071329 is a cis-eQTL, with the G/G genotype conferring enhanced PPARGC1B expression, with consequent improved mitochondrial function and response to norepinephrine in brown adipocytes. This genetic variant, and as yet undetermined eQTLs, at PPARGC1B could prove useful in genotype-based precision medicine for obesity treatment.

A genome-wide association study of social trust in 33,882 Danish blood donors.

Social trust is a heritable trait that has been linked with physical health and longevity. In this study, we performed genome-wide association studies of self-reported social trust in n = 33,882 Danish blood donors. We observed genome-wide and local evidence of genetic similarity with other brain-related phenotypes and estimated the single nucleotide polymorphism-based heritability of trust to be 6% (95% confidence interval = (2.1, 9.9)). In our discovery cohort (n = 25,819), we identified one significantly associated locus (lead variant: rs12776883) in an intronic enhancer region of PLPP4, a gene highly expressed in brain, kidneys, and testes. However, we could not replicate the signal in an independent set of donors who were phenotyped a year later (n = 8063). In the subsequent meta-analysis, we found a second significantly associated variant (rs71543507) in an intergenic enhancer region. Overall, our work confirms that social trust is heritable, and provides an initial look into the genetic factors that influence it.

Reduced Expression Level of Protein Phosphatase PPM1E Serves to Maintain Insulin Secretion in Type 2 Diabetes.

Reversible phosphorylation is an important regulatory mechanism. Regulation of protein phosphorylation in ß-cells has been extensively investigated, but less is known about protein dephosphorylation. To understand the role of protein dephosphorylation in ß-cells and type 2 diabetes (T2D), we first examined mRNA expression of the type 2C family (PP2C) of protein phosphatases in islets from T2D donors. Phosphatase expression overall was changed in T2D, and that of PPM1E was the most markedly downregulated. PPM1E expression correlated inversely with HbA1c. Silencing of PPM1E increased glucose-stimulated insulin secretion (GSIS) in INS-1 832/13 cells and/or islets from patients with T2D, whereas PPM1E overexpression decreased GSIS. Increased GSIS after PPM1E silencing was associated with decreased oxidative stress, elevated cytosolic Ca2+ levels and ATP to ADP ratio, increased hyperpolarization of the inner mitochondrial membrane, and phosphorylation of CaMKII, AMPK, and acetyl-CoA carboxylase. Silencing of PPM1E, however, did not change insulin content. Increased GSIS, cell viability, and activation of AMPK upon metformin treatment in ß-cells were observed upon PPM1E silencing. Thus, protein dephosphorylation via PPM1E abrogates GSIS. Consequently, reduced PPM1E expression in T2D may be a compensatory response of ß-cells to uphold insulin secretion under metabolic duress. Targeting PPM1E in ß-cells may thus represent a novel therapeutic strategy for treatment of T2D.

Identification of a weight loss-associated causal eQTL in MTIF3 and the effects of MTIF3 deficiency on human adipocyte function.

Genetic variation at the MTIF3 (Mitochondrial Translational Initiation Factor 3) locus has been robustly associated with obesity in humans, but the functional basis behind this association is not known. Here, we applied luciferase reporter assay to map potential functional variants in the haplotype block tagged by rs1885988 and used CRISPR-Cas9 to edit the potential functional variants to confirm the regulatory effects on MTIF3 expression. We further conducted functional studies on MTIF3-deficient differentiated human white adipocyte cell line (hWAs-iCas9), generated through inducible expression of CRISPR-Cas9 combined with delivery of synthetic MTIF3-targeting guide RNA. We demonstrate that rs67785913-centered DNA fragment (in LD with rs1885988, r2 > 0.8) enhances transcription in a luciferase reporter assay, and CRISPR-Cas9-edited rs67785913 CTCT cells show significantly higher MTIF3 expression than rs67785913 CT cells. Perturbed MTIF3 expression led to reduced mitochondrial respiration and endogenous fatty acid oxidation, as well as altered expression of mitochondrial DNA-encoded genes and proteins, and disturbed mitochondrial OXPHOS complex assembly. Furthermore, after glucose restriction, the MTIF3 knockout cells retained more triglycerides than control cells. This study demonstrates an adipocyte function-specific role of MTIF3, which originates in the maintenance of mitochondrial function, providing potential explanations for why MTIF3 genetic variation at rs67785913 is associated with body corpulence and response to weight loss interventions.

A phenome-wide comparative analysis of genetic discordance between obesity and type 2 diabetes.

Obesity and type 2 diabetes are causally related, yet there is considerable heterogeneity in the consequences of both conditions and the mechanisms of action are poorly defined. Here we show a genetic-driven approach defining two obesity profiles that convey highly concordant and discordant diabetogenic effects. We annotate and then compare association signals for these profiles across clinical and molecular phenotypic layers. Key differences are identified in a wide range of traits, including cardiovascular mortality, fat distribution, liver metabolism, blood pressure, specific lipid fractions and blood levels of proteins involved in extracellular matrix remodelling. We find marginal differences in abundance of Bacteroidetes and Firmicutes bacteria in the gut. Instrumental analyses reveal prominent causal roles for waist-to-hip ratio, blood pressure and cholesterol content of high-density lipoprotein particles in the development of diabetes in obesity. We prioritize 17 genes from the discordant signature that convey protection against type 2 diabetes in obesity, which may represent logical targets for precision medicine approaches.

The streptococcal collagen-binding protein CNE specifically interferes with alphaVbeta3-mediated cellular interactions with triple helical collagen.

Collagen fibers expose distinct domains allowing for specific interactions with other extracellular matrix proteins and cells. To investigate putative collagen domains that govern integrin α(V)ß(3)-mediated cellular interactions with native collagen fibers we took advantage of the streptococcal protein CNE that bound native fibrillar collagens. CNE specifically inhibited α(V)ß(3)-dependent cell-mediated collagen gel contraction, PDGF BB-induced and α(V)ß(3)-mediated adhesion of cells, and binding of fibronectin to native collagen. Using a Toolkit composed of overlapping, 27-residue triple helical segments of collagen type II, two CNE-binding sites present in peptides II-1 and II-44 were identified. These peptides lack the major binding site for collagen-binding ß(1) integrins, defined by the peptide GFOGER. Peptide II-44 corresponds to a region of collagen known to bind collagenases, discoidin domain receptor 2, SPARC (osteonectin), and fibronectin. In addition to binding fibronectin, peptide II-44 but not II-1 inhibited α(V)ß(3)-mediated collagen gel contraction and, when immobilized on plastic, supported adhesion of cells. Reduction of fibronectin expression by siRNA reduced PDGF BB-induced α(V)ß(3)-mediated contraction. Reconstitution of collagen types I and II gels in the presence of CNE reduced collagen fibril diameters and fibril melting temperatures. Our data indicate that contraction proceeded through an indirect mechanism involving binding of cell-produced fibronectin to the collagen fibers. Furthermore, our data show that cell-mediated collagen gel contraction does not directly depend on the process of fibril formation.

Vaccination against the extra domain-B of fibronectin as a novel tumor therapy.

Monoclonal antibody-based therapies have made an important contribution to current treatment strategies for cancer and autoimmune disease. However, the cost for these new drugs puts a significant strain on the health-care economy, resulting in limited availability for patients. Therapeutic vaccination, defined as induction of immunity against a disease-related self-molecule, is therefore an attractive alternative. To analyze the potential of such an approach, we have developed a vaccine against the extra domain-B (ED-B) of fibronectin. This 91-aa domain, inserted by alternative splicing, is expressed during vasculogenesis in the embryo, but essentially undetectable under normal conditions in the adult. However, ED-B is highly expressed around angiogenic vasculature, such as in tumorigenesis. Here, we demonstrate that it is possible to break self-tolerance and induce a strong antibody response against ED-B by vaccination. Nineteen of 20 vaccinated mice responded with production of anti-ED-B antibodies and displayed a 70% reduction in tumor size compared to those lacking anti-ED-B antibodies. Analysis of the tumor tissue revealed that immunization against ED-B induced several changes, consistent with an attack by the immune system. These data show that tumor vascular antigens are highly interesting candidates for development of therapeutic vaccines targeting solid tumors.

Type I interferon system activation and association with disease manifestations in systemic sclerosis.

OBJECTIVES: To study the presence of interferogenic autoantibodies in systemic sclerosis (SSc) and their correlation with clinical manifestations, serum levels of interferon alpha (IFNalpha) and chemokines of importance in the disease process. METHODS: Peripheral blood mononuclear cells (PBMCs) or purified plasmacytoid dendritic cells (pDCs) from healthy donors were stimulated with sera from patients with SSc (n=70) or healthy individuals (n=30), together with necrotic or apoptotic cell material. The IFNalpha produced and serum levels of IFNalpha, IFN-inducible protein-10 (IP-10)/chemokine (C-X-C motif) ligand 10, monocyte chemoattractant protein-1 (MCP-1)/(C-C motif) ligand-2 (CCL-2), macrophage inflammatory protein-1alpha (MIP-1alpha)/CCL-3 and RANTES/CCL-5 were measured and correlated with the presence of autoantibodies and clinical manifestations in the patients with SSc. RESULTS: Sera from both diffuse SSc and limited SSc contained interferogenic antibodies, which correlated with the presence of anti-ribonucleoprotein and anti-Sjögren syndrome antigen autoantibodies. The pDCs were responsible for the IFNalpha production which required interaction with FcgammaRII and endocytosis. Increased serum levels of IP-10 were associated with vascular manifestations such as cardiac involvement (p=0.027) and pulmonary arterial hypertension (p=0.036). Increased MCP-1 or IFNalpha serum levels were associated with lung fibrosis (p=0.019 and 0.048, respectively). Digital ulcers including digital loss were associated with increased serum levels of IFNalpha (p=0.029). CONCLUSION: An activated type I IFN system previously seen in several other systemic autoimmune diseases is also present in SSc and may contribute to the vascular pathology and affect the profibrotic process.

Asporin competes with decorin for collagen binding, binds calcium and promotes osteoblast collagen mineralization.

The interactions of the ECM (extracellular matrix) protein asporin with ECM components have previously not been investigated. Here, we show that asporin binds collagen type I. This binding is inhibited by recombinant asporin fragment LRR (leucine-rich repeat) 10-12 and by full-length decorin, but not by biglycan. We demonstrate that the polyaspartate domain binds calcium and regulates hydroxyapatite formation in vitro. In the presence of asporin, the number of collagen nodules, and mRNA of osteoblastic markers Osterix and Runx2, were increased. Moreover, decorin or the collagen-binding asporin fragment LRR 10-12 inhibited the pro-osteoblastic activity of full-length asporin. Our results suggest that asporin and decorin compete for binding to collagen and that the polyaspartate in asporin directly regulates collagen mineralization. Therefore asporin has a role in osteoblast-driven collagen biomineralization activity. We also show that asporin can be expressed in Escherichia coli (Rosetta-gami) with correctly positioned cysteine bridges, and a similar system can possibly be used for the expression of other SLRPs (small LRR proteoglycans/proteins).

Cathepsin g Degrades Both Glycosylated and Unglycosylated Regions of Lubricin, a Synovial Mucin.

Lubricin (PRG4) is a mucin type protein that plays an important role in maintaining normal joint function by providing lubrication and chondroprotection. Improper lubricin modification and degradation has been observed in idiopathic osteoarthritis (OA), while the detailed mechanism still remains unknown. We hypothesized that the protease cathepsin G (CG) may participate in degrading lubricin in synovial fluid (SF). The presence of endogenous CG in SF was confirmed in 16 patients with knee OA. Recombinant human lubricin (rhPRG4) and native lubricin purified from the SF of patients were incubated with exogenous CG and lubricin degradation was monitored using western blot, staining by Coomassie or Periodic Acid-Schiff base in gels, and with proteomics. Full length lubricin (∼300 kDa), was efficiently digested with CG generating a 25-kDa protein fragment, originating from the densely glycosylated mucin domain (∼250 kDa). The 25-kDa fragment was present in the SF from OA patients, and the amount was increased after incubation with CG. A CG digest of rhPRG4 revealed 135 peptides and 72 glycopeptides, and confirmed that the protease could cleave in all domains of lubricin, including the mucin domain. Our results suggest that synovial CG may take part in the degradation of lubricin, which could affect the pathological decrease of the lubrication in degenerative joint disease.