Publications by authors named "John S Mort"

46 Publications

Collagen type I degradation fragments act through the collagen receptor LAIR-1 to provide a negative feedback for osteoclast formation.

Bone 2018 12 12;117:23-30. Epub 2018 Sep 12.

Faculty of Dentistry, McGill University, Montreal, Quebec, Canada; Shriners Hospital for Children-Canada, Montreal, Quebec H3G 1A6, Canada. Electronic address:

The major organic component of bone is collagen type I. Osteoclasts are terminally differentiated multinucleated cells of hematopoietic origin that are essential for physiological development of bone and teeth. We examined if osteoclast differentiation from murine bone marrow precursors is affected by collagen type I, or by its degradation products produced by human recombinant cathepsin K. Osteoclasts formation was dose-dependently inhibited in the presence of full length collagen type I or its 30-75 kDa degradation products added to the osteoclast differentiation media for the duration of an experiment. Collagen degradation fragments signaled through SH-2 phosphatases, inhibiting calcium signaling and NFATc1 translocation in osteoclast precursors. Osteoclasts and their precursors expressed a collagen receptor of leukocyte receptor complex family, LAIR-1. Importantly, collagen fragments failed to inhibit osteoclast formation from LAIR-1 deficient murine osteoclast precursors. This study demonstrates that collagen degradation fragments inhibit osteoclast formation acting through LAIR-1, providing a novel mechanism for the physiologically-relevant negative control of osteoclastogenesis.
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http://dx.doi.org/10.1016/j.bone.2018.09.006DOI Listing
December 2018

Aggrecan heterogeneity in articular cartilage from patients with osteoarthritis.

BMC Musculoskelet Disord 2016 Feb 18;17:89. Epub 2016 Feb 18.

Research Unit, Shriners Hospital for Children, 1003, boul. Décarie, Montreal, Quebec, H4A 0A9, Canada.

Background: Aggrecan degradation is the hallmark of cartilage degeneration in osteoarthritis (OA), though it is unclear whether a common proteolytic process occurs in all individuals.

Methods: Aggrecan degradation in articular cartilage from the knees of 33 individuals with OA, who were undergoing joint replacement surgery, was studied by immunoblotting of tissue extracts.

Results: Matrix metalloproteinases (MMPs) and aggrecanases are the major proteases involved in aggrecan degradation within the cartilage, though the proportion of aggrecan cleavage attributable to MMPs or aggrecanases was variable between individuals. However, aggrecanases were more associated with the increase in aggrecan loss associated with OA than MMPs. While the extent of aggrecan cleavage was highly variable between individuals, it was greatest in areas of cartilage adjacent to sites of cartilage erosion compared to sites more remote within the same joint. Analysis of link protein shows that in some individuals additional proteolytic mechanisms must also be involved to some extent.

Conclusions: The present studies indicate that there is no one protease, or a fixed combination of proteases, responsible for cartilage degradation in OA. Thus, rather than targeting the individual proteases for OA therapy, directing research to techniques that control global protease generation may be more productive.
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http://dx.doi.org/10.1186/s12891-016-0944-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4758135PMC
February 2016

Reduction in disease progression by inhibition of transforming growth factor α-CCL2 signaling in experimental posttraumatic osteoarthritis.

Arthritis Rheumatol 2015 Oct;67(10):2691-701

Western University Schulich School of Medicine and Dentistry and Children's Health Research Institute, London, Ontario, Canada.

Objective: Transforming growth factor α (TGFα) is increased in osteoarthritic (OA) cartilage in rats and humans and modifies chondrocyte phenotype. CCL2 is increased in OA cartilage and stimulates proteoglycan loss. This study was undertaken to test whether TGFα and CCL2 cooperate to promote cartilage degradation and whether inhibiting either reduces disease progression in a rat model of posttraumatic OA.

Methods: Microarray analysis was used to profile expression of messenger RNA (mRNA) for Tgfa, Ccl2, and related genes in a rat model of posttraumatic OA. Rat primary chondrocytes and articular cartilage explants were treated with TGFα in the presence or absence of MEK-1/2, p38, phosphatidylinositol 3-kinase, Rho-associated protein kinase, or CCR2 inhibitors and immunostained for markers of cartilage degradation. The rat model was used to administer pharmacologic inhibitors of TGFα (AG1478) and CCL2 (RS504393) signaling for up to 10 weeks and assess histopathology and serum biomarkers of cartilage synthesis (C-propeptide of type II collagen [CPII]) and breakdown (C2C).

Results: Tgfa and Ccl2 mRNA were simultaneously up-regulated in articular cartilage in the rat model of posttraumatic OA. TGFα induced expression of CCL2, Mmp3, and Tnf in primary chondrocytes. Cleavage of type II collagen and aggrecan (by matrix metalloproteinases and ADAMTS-4/5, respectively) induced by TGFα was blocked by pharmacologic inhibition of CCL2 in cartilage explants. In vivo pharmacologic inhibition of TGFα or CCL2 signaling reduced Osteoarthritis Research Society International cartilage histopathology scores and increased serum CPII levels, but only TGFα inhibition reduced C2C levels intreated versus untreated rat OA cartilage.

Conclusion: TGFα signaling stimulates cartilage degradation via a CCL2-dependent mechanism, but pharmacologic inhibition of the TGFα-CCL2 axis reduces experimental posttraumatic OA progression in vivo.
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http://dx.doi.org/10.1002/art.39255DOI Listing
October 2015

Recessive osteogenesis imperfecta caused by missense mutations in SPARC.

Am J Hum Genet 2015 Jun 28;96(6):979-85. Epub 2015 May 28.

Shriners Hospital for Children, Montréal, QC H3G 1A6, Canada. Electronic address:

Secreted protein, acidic, cysteine-rich (SPARC) is a glycoprotein that binds to collagen type I and other proteins in the extracellular matrix. Using whole-exome sequencing to identify the molecular defect in two unrelated girls with severe bone fragility and a clinical diagnosis of osteogenesis imperfecta type IV, we identified two homozygous variants in SPARC (GenBank: NM_003118.3; c.497G>A [p.Arg166His] in individual 1; c.787G>A [p.Glu263Lys] in individual 2). Published modeling and site-directed mutagenesis studies had previously shown that the residues substituted by these mutations form an intramolecular salt bridge in SPARC and are essential for the binding of SPARC to collagen type I. The amount of SPARC secreted by skin fibroblasts was reduced in individual 1 but appeared normal in individual 2. The migration of collagen type I alpha chains produced by these fibroblasts was mildly delayed on SDS-PAGE gel, suggesting some overmodification of collagen during triple helical formation. Pulse-chase experiments showed that collagen type I secretion was mildly delayed in skin fibroblasts from both individuals. Analysis of an iliac bone sample from individual 2 showed that trabecular bone was hypermineralized on the material level. In conclusion, these observations show that homozygous mutations in SPARC can give rise to severe bone fragility in humans.
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http://dx.doi.org/10.1016/j.ajhg.2015.04.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4457955PMC
June 2015

Repeated exposure to high-frequency low-amplitude vibration induces degeneration of murine intervertebral discs and knee joints.

Arthritis Rheumatol 2015 May;67(8):2164-75

University of Western Ontario Schulich School of Medicine and Dentistry, London, Ontario, Canada.

Objective: High-frequency, low-amplitude whole-body vibration (WBV) is being used to treat a range of musculoskeletal disorders; however, there is surprisingly limited knowledge regarding its effect(s) on joint tissues. This study was undertaken to examine the effects of repeated exposure to WBV on bone and joint tissues in an in vivo mouse model.

Methods: Ten-week-old male mice were exposed to vertical sinusoidal vibration under conditions that mimic those used clinically in humans (30 minutes per day, 5 days per week, at 45 Hz with peak acceleration at 0.3g). Following WBV, skeletal tissues were examined by micro-computed tomography, histologic analysis, and immunohistochemistry, and gene expression was quantified using real-time polymerase chain reaction.

Results: Following 4 weeks of WBV, intervertebral discs showed histologic hallmarks of degeneration in the annulus fibrosus, disruption of collagen organization, and increased cell death. Greater Mmp3 expression in the intervertebral disc, accompanied by enhanced collagen and aggrecan degradation, was found in mice exposed to WBV as compared to controls. Examination of the knee joints after 4 weeks of WBV revealed meniscal tears and focal damage to the articular cartilage, changes resembling osteoarthritis. Moreover, mice exposed to WBV also demonstrated greater Mmp13 gene expression and enhanced matrix metalloproteinase-mediated collagen and aggrecan degradation in articular cartilage as compared to controls. No changes in trabecular bone microarchitecture or density were detected in the proximal tibia.

Conclusion: Our experiments reveal significant negative effects of WBV on joint tissues in a mouse model. These findings suggest the need for future studies of the effects of WBV on joint health in humans.
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http://dx.doi.org/10.1002/art.39154DOI Listing
May 2015

Cole-Carpenter syndrome is caused by a heterozygous missense mutation in P4HB.

Am J Hum Genet 2015 Mar 12;96(3):425-31. Epub 2015 Feb 12.

Shriners Hospital for Children, Montréal, QC H3G 1A6, Canada.

Cole-Carpenter syndrome is a severe bone fragility disorder that is characterized by frequent fractures, craniosynostosis, ocular proptosis, hydrocephalus, and distinctive facial features. To identify the cause of Cole-Carpenter syndrome in the two individuals whose clinical results were presented in the original description of this disorder, we performed whole-exome sequencing of genomic DNA samples from both individuals. The two unrelated individuals had the same heterozygous missense mutation in exon 9 of P4HB (NM_000918.3: c.1178A>G [p.Tyr393Cys]), the gene that encodes protein disulfide isomerase (PDI). In one individual, the P4HB mutation had arisen de novo, whereas in the other the mutation was transmitted from the clinically unaffected father who was a mosaic carrier of the variant. The mutation was located in the C-terminal disulfide isomerase domain of PDI, sterically close to the enzymatic center, and affected disulfide isomerase activity in vitro. Skin fibroblasts showed signs of increased endoplasmic reticulum stress, but despite the reported importance of PDI for collagen type I production, the rate of collagen type I secretion appeared normal. In conclusion, Cole-Carpenter syndrome is caused by a specific de novo mutation in P4HB that impairs the disulfide isomerase activity of PDI.
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http://dx.doi.org/10.1016/j.ajhg.2014.12.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4375435PMC
March 2015

The role of aggrecan in normal and osteoarthritic cartilage.

J Exp Orthop 2014 Dec 16;1(1). Epub 2014 Jul 16.

Shriners Hospital for Children and McGill University, Montreal, Quebec, Canada.

Aggrecan is a large proteoglycan bearing numerous chondroitin sulfate and keratan sulfate chains that endow articular cartilage with its ability to withstand compressive loads. It is present in the extracellular matrix in the form of proteoglycan aggregates, in which many aggrecan molecules interact with hyaluronan and a link protein stabilizes each interaction. Aggrecan structure is not constant throughout life, but changes due to both synthetic and degradative events. Changes due to synthesis alter the structure of the chondroitin sulfate and keratan sulfate chains, whereas those due to degradation cause cleavage of all components of the aggregate. These latter changes can be viewed as being detrimental to cartilage function and are enhanced in osteoarthritic cartilage, resulting in aggrecan depletion and predisposing to cartilage erosion. Matrix metalloproteinases and aggrecanases play a major role in aggrecan degradation and their production is upregulated by mediators associated with joint inflammation and overloading. The presence of increased levels of aggrecan fragments in synovial fluid has been used as a marker of ongoing cartilage destruction in osteoarthritis. During the early stages of osteoarthritis it may be possible to retard the destructive process by enhancing the production of aggrecan and inhibiting its degradation. Aggrecan production also plays a central role in cartilage repair techniques involving stem cell or chondrocyte implantation into lesions. Thus aggrecan participates in both the demise and survival of articular cartilage.
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http://dx.doi.org/10.1186/s40634-014-0008-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4648834PMC
December 2014

Cells from the skin of patients with systemic sclerosis secrete chitinase 3-like protein 1.

BBA Clin 2014 Jun 8;1:2-11. Epub 2014 Jan 8.

Shriners Hospital for Children, Department of Surgery, McGill University, 1529 Cedar Avenue, Montréal, Quebec H3G 1A6, Canada.

Background: The chitinase-like protein, Chi3L1, is associated with increased fibrotic activity as well as inflammatory processes. The capacity of skin cells from systemic sclerosis (SSc) patients to produce Chi3L1, and the stimulation of its synthesis by cytokines or growth factors known to be associated with SSc, was investigated.

Methods: Cells were isolated from forearm and/or abdomen skin biopsies taken from SSc patients and normal individuals and stimulated with cytokines and growth factors to assess Chi3L1 expression. Chi3L1-expressing cells were characterized by immunohistochemical staining.

Results: Chi3L1 was not secreted by skin cells from normal individuals nor was its synthesis induced by any of the cytokines or growth factors investigated. In contrast, Chi3L1 secretion was induced by OSM or IL-1 in cells from all forearm biopsies of SSc patients, and endogenous secretion in the absence of cytokines was detected in several specimens. Patients with Chi3L1-producing cells at both the arm and abdomen had a disease duration of less than 3 years. Endogenous Chi3L1 production was not a property of the major fibroblast population nor of myofibroblasts, but rather was related to the presence of stem-like cells not present in normal skin. Other cells, however, contributed to the upregulation of Chi3L1 by OSM.

Conclusions: The emergence of cells primed to respond to OSM with increased Chi3L1 production appears to be associated with pathological processes active in SSc.

General Significance: The presence of progenitor cells expressing the chilectin Chi3L1 in SSc skin appears to play a role in the initiation of the disease process.
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http://dx.doi.org/10.1016/j.bbacli.2013.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4633946PMC
June 2014

Efficient processing of procathepsin K to the mature form.

Protein Expr Purif 2013 Sep 8;91(1):37-41. Epub 2013 Jul 8.

Genetics Unit, Shriners Hospital for Children and Department of Surgery, McGill University, Montreal, Quebec, Canada.

The proteolysis of collagen fibrils by cathepsin K is a hallmark of bone catabolism and tissue degeneration. The production of active recombinant cathepsin K is central for our ability to study the mechanisms by which these processes occur. Here we report an efficient processing method for the preparation of recombinant cathepsin K expressed in Pichia pastoris. Methanol precipitation of crude media and autoactivation in the absence of a reducing agent allows for the reversible inhibition of the enzyme prior to subsequent purification steps. The resultant purified enzyme is both resistant to autolysis and effective at cleaving collagen.
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http://dx.doi.org/10.1016/j.pep.2013.06.012DOI Listing
September 2013

Increased type II collagen cleavage by cathepsin K and collagenase activities with aging and osteoarthritis in human articular cartilage.

Arthritis Res Ther 2012 May 14;14(3):R113. Epub 2012 May 14.

Genetics Unit, Shriners Hospitals for Children, 1529 Cedar Avenue, Montreal, QC H3G 1A6, Canada.

Introduction: The intra-helical cleavage of type II collagen by proteases, including collagenases and cathepsin K, is increased with aging and osteoarthritis (OA) in cartilage as determined by immunochemical assays. The distinct sites of collagen cleavage generated by collagenases and cathepsin K in healthy and OA human femoral condylar cartilages were identified and compared.

Methods: Fixed frozen cartilage sections were examined immunohistochemically, using antibodies that react with the collagenase-generated cleavage neoepitopes, C2C and C1,2C, and the primary cleavage neoepitope (C2K) generated in type II collagen by the action of cathepsin K and possibly by other proteases, but not by any collagenases studied to date.

Results: In most cases, the staining patterns for collagen cleavage were similar for all three epitopes: weak to moderate mainly pericellular staining in non-OA cartilage from younger individuals and stronger, more widespread staining in aging and OA cartilages that often extended from the superficial to the mid/deep zone of the tissue. In very degenerate OA specimens, with significant disruption of the articular surface, staining was distributed throughout most of the cartilage matrix.

Conclusions: Cleavage of collagen by proteases usually arises pericellularly around chondrocytes at and near the articular surface, subsequently becoming more intense and extending progressively deeper into the cartilage with aging and OA. The close correspondence between the distributions of these products suggests that both collagenases and cathepsin K, and other proteases that may generate this distinct cathepsin K cleavage site, are usually active in the same sites in the degradation of type II collagen.
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http://dx.doi.org/10.1186/ar3839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3446490PMC
May 2012

Analysis of aggrecan catabolism by immunoblotting and immunohistochemistry.

Methods Mol Biol 2012 ;836:219-37

Research Unit, Shriners Hospital for Children, Montreal, QC, Canada.

Aggrecan is essential for the normal function of articular cartilage and intervertebral disc, where it provides the ability for the tissues to withstand compressive loading. This property depends on both the high charge density endowed by its numerous chondroitin sulfate and keratan sulfate chains and its ability to form large molecular aggregates via interaction with hyaluronan. Degradation of aggrecan via the action of proteases takes place throughout life and the degradation products accumulate in the tissue and impair its function. Such degradation is exacerbated in degenerative or inflammatory joint disorders. The use of antibodies recognizing the various regions of aggrecan and the neoepitopes generated upon proteolytic cleavage has shown that matrix metalloproteinases and aggrecanases, members of the ADAMTS family, are responsible for aggrecan degradation, both throughout life and in disease. By using immunoblotting techniques, it is possible to determine the extent of aggrecan degradation and to identify the degradation products that have accumulated in the tissue, and immunohistochemistry allows the location of the aggrecan degradation to be established.
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http://dx.doi.org/10.1007/978-1-61779-498-8_15DOI Listing
May 2012

Alarmins S100A8 and S100A9 elicit a catabolic effect in human osteoarthritic chondrocytes that is dependent on Toll-like receptor 4.

Arthritis Rheum 2012 May;64(5):1477-87

Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

Objective: S100A8 and S100A9 are two Ca(2+) binding proteins classified as damage-associated molecular patterns or alarmins that are found in high amounts in the synovial fluid of osteoarthritis (OA) patients. The purpose of this study was to investigate whether S100A8 and/or S100A9 can interact with chondrocytes from OA patients to increase catabolic mediators.

Methods: Using immunohistochemistry, we stained for S100A8 and S100A9 protein, matrix metalloproteinases (MMPs), and a cartilage-breakdown epitope specific for MMPs (VDIPEN) in cartilage from OA donors. Isolated chondrocytes or explants from OA and non-OA donors were stimulated with S100A8 and/or S100A9. Messenger RNA and protein levels of MMPs, cytokines, and cartilage matrix molecules were determined with quantitative reverse transcription-polymerase chain reaction and Luminex techniques, respectively. For receptor blocking studies, specific inhibitors for Toll-like receptor 4 (TLR-4), receptor for advanced glycation end products (RAGE), and carboxylated glycans were used.

Results: In cartilage from OA patients, the expression of S100A8 and S100A9 protein close to chondrocytes was associated with proteoglycan depletion and expression of MMP-1, MMP-3, and VDIPEN. Stimulation of chondrocytes with S100A8 and S100A9 caused a strong up-regulation of catabolic markers (MMPs 1, 3, 9, and 13, interleukin-6 [IL-6], IL-8, and monocyte chemotactic protein 1) and down-regulation of anabolic markers (aggrecan and type II collagen), thereby favoring cartilage breakdown. Blocking TLR-4, but not carboxylated glycans or RAGE, inhibited the S100 effect. The catabolic S100 effect was significantly more pronounced in chondrocytes from OA patients as compared to those from non-OA patients, possibly due to higher TLR-4 expression.

Conclusion: S100A8 and S100A9 have a catabolic effect on human chondrocytes that is TLR-4 dependent. OA chondrocytes are more sensitive than normal chondrocytes to S100 stimulation.
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http://dx.doi.org/10.1002/art.33495DOI Listing
May 2012

Partially redundant functions of Adamts1 and Adamts4 in the perinatal development of the renal medulla.

Dev Dyn 2011 Jul 16;240(7):1806-14. Epub 2011 May 16.

Départment de Biomédecine Vétérinaire, Université de Montréal, St-Hyacinthe, Québec, Canada.

Adamts4 encodes a widely-expressed proteinase thought to be involved in processes ranging from cartilage metabolism to ovarian follicle development. To study its physiological roles, Adamts4-null mice were created by gene targeting. Unexpectedly, these were found to be phenotypically normal, suggesting that other gene(s) may compensate for its loss. Adamts4(-/-) mice were, therefore, crossed with a strain lacking Adamts1, whose pattern of expression and substrate specificity overlap that of Adamts4. Most (>95%) Adamts1(-/-) ;Adamts4(-/-) mice died within 72 hr after birth with a marked thinning of the renal medulla. The renal defect was not observed in embryonic Adamts1(-/-) ;Adamts4(-/-) kidneys, but became apparent around birth. The few (<5%) Adamts1(-/-) ;Adamts4(-/-) animals to reach adulthood had the same renal phenotype seen in newborns. This study is thus the first to report Adamts4 expression and function in the mammalian kidney, and to demonstrate that Adamts1 and Adamts4 play redundant and essential roles in perinatal kidney development.
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http://dx.doi.org/10.1002/dvdy.22662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3116038PMC
July 2011

Structural basis for the recognition and cleavage of histone H3 by cathepsin L.

Nat Commun 2011 Feb 15;2:197. Epub 2011 Feb 15.

Structural Genomics Consortium, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada.

Proteolysis of eukaryotic histone tails has emerged as an important factor in the modulation of cell-cycle progression and cellular differentiation. The recruitment of lysosomal cathepsin L to the nucleus where it mediates proteolysis of the mouse histone H3 tail has been described recently. Here, we report the three-dimensional crystal structures of a mature, inactive mutant of human cathepsin L alone and in complex with a peptide derived from histone H3. Canonical substrate-cathepsin L interactions are observed in the complex between the protease and the histone H3 peptide. Systematic analysis of the impact of posttranslational modifications at histone H3 on substrate selectivity suggests cathepsin L to be highly accommodating of all modified peptides. This is the first report of cathepsin L-histone H3 interaction and the first structural description of cathepsin L in complex with a substrate.
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http://dx.doi.org/10.1038/ncomms1204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3105313PMC
February 2011

MMPs are less efficient than ADAMTS5 in cleaving aggrecan core protein.

Matrix Biol 2011 Mar 3;30(2):145-53. Epub 2010 Nov 3.

Genetics Unit, Shriners Hospital for Children, Montreal, Canada.

Aggrecan degradation in articular cartilage occurs predominantly through proteolysis and has been attributed to the action of members of the matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) families. Both families of enzymes cleave aggrecan at specific sites within the aggrecan core protein. One cleavage site within the interglobular domain (IGD), between Glu(373-374)Ala and five additional sites in the chondroitin sulfate-2 (CS-2) region of aggrecan were characterized as "aggrecanase" (ADAMTS) cleavage sites, while cleavage between Ser(341-342)Phe within the IGD of bovine aggrecan is attributed to MMP action. The objective of this study was to assess the cleavage efficiency of MMPs relative to ADAMTS and their contribution to aggrecan proteolysis in vitro. The analysis of aggrecan IGD degradation in bovine articular cartilage explants treated with catabolic cytokines over a 19-day period showed that MMP-mediated degradation of aggrecan within the IGD can only be observed following day 12 of culture. This delay is associated with the lack of activation of proMMPs during the first 12 days of culture. Analysis of MMP1, 2, 3, 7, 8, 9, 12, 13 and ADAMTS5 efficiencies at cleaving within the aggrecan IGD and CS-2 region in vitro was carried out by the digestion of bovine aggrecan with the various enzymes and Western blot analysis using aggrecan anti-G1 and anti-G3 antibodies. Of these MMPs, MMP12 was the most efficient at cleaving within the aggrecan IGD. In addition to cleavage in the IGD, MMP, 3, 7, 8 and 12 were also able to degrade the aggrecan CS-2 region. MMP3 and MMP12 were able to degrade aggrecan at the very C-terminus of the CS-2 region, cleaving the Glu(2047-2048)Ala bond which was previously shown to be cleaved by ADAMTS5. However, in comparison to ADAMTS5, MMP3 was about 100 times and 10 times less efficient at cleaving within the aggrecan IGD and CS-2 regions, respectively. Collectively, our results showed that the delayed activation of proMMPs and the relatively low cleavage efficiency of MMPs can explain the minor contribution of these enzymes to aggrecan catabolism in vivo. This study also uncovered a potential role for MMPs in the C-terminal truncation of aggrecan.
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http://dx.doi.org/10.1016/j.matbio.2010.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3057330PMC
March 2011

Rho/ROCK and MEK/ERK activation by transforming growth factor-alpha induces articular cartilage degradation.

Lab Invest 2010 Jan 12;90(1):20-30. Epub 2009 Oct 12.

CIHR Group in Skeletal Development and Remodeling, London, ON, Canada.

Identification and characterization of therapeutic targets for joint conditions, such as osteoarthritis (OA), is exceedingly important for addressing the increasing burden of disease. Transforming growth factor-alpha (TGFalpha) is upregulated by articular chondrocytes in experimentally induced and human OA. To test the potential involvement of TGFalpha, which is an activator of epidermal growth factor receptor (EGFR) signaling, in joint degeneration and to identify signaling mechanisms mediating articular chondrocyte responses to TGFalpha, rat chondrocytes and osteochondral explants were treated with TGFalpha and various inhibitors of intracellular signaling pathways. Stimulation of EGFR signaling in articular chondrocytes by TGFalpha resulted in the activation of RhoA/ROCK (Rho kinase), MEK (MAPK/ERK kinase)/ERK (extracellular-signal-regulated kinase), PI3K (phosphoinositide 3-kinase) and p38 MAPK (mitogen-activated protein kinase) pathways. Modification of the chondrocyte actin cytoskeleton was stimulated by TGFalpha, but inhibition of only Rho or ROCK activation prevented morphological changes. TGFalpha suppressed expression of anabolic genes including Sox9, type II collagen and aggrecan, which were rescued only by inhibiting MEK/ERK activation. Furthermore, catabolic factor upregulation by TGFalpha was prevented by ROCK and p38 MAPK inhibition, including matrix metalloproteinase-13 and tumor necrosis factor-alpha, which are well known to contribute to cartilage digestion in OA. To assess the ability of TGFalpha to stimulate degradation of mature articular cartilage, type II collagen and aggrecan cleavage fragments were analyzed in rat osteochondral explants exposed to exogenous TGFalpha. Normal articular cartilage contained low levels of both cleavage fragments, but high levels were observed in the cartilage treated with TGFalpha. Selective inhibition of MEK/ERK and Rho/ROCK activation greatly reduced or completely prevented excess type II collagen and aggrecan degradation in response to TGFalpha. These data suggest that TGFalpha is a strong stimulator of cartilage degradation and that Rho/ROCK and MEK/ERK signaling have critical roles in mediating these effects.
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http://dx.doi.org/10.1038/labinvest.2009.111DOI Listing
January 2010

Neoepitopes reveal the features of type II collagen cleavage and the identity of a collagenase involved in the transformation of the epiphyses anlagen in development.

Dev Dyn 2009 Jun;238(6):1547-63

Genetics Unit, Shriners Hospital for Children, Montreal, Quebec, Canada.

In long bone development, the evolution of the cartilaginous anlagen into a secondary ossification center is initiated by the formation of canals. The excavation to create the canals is achieved through lysis of the two major cartilage components, aggrecan, and the type II collagen (COL2) fibril. The present study examines the lysis of the fibril. Because it is known that matrix metalloproteinases (MMPs) cleave COL2 in vitro at the Gly(775)-Leu(776) bond, it has been reasoned that, if such cleavage is detected in relation to the canals, it can be concluded that a collagenase is involved. Furthermore, because MMPs undergo change in domain structure with activation resulting in propeptide domain loss then, if such a loss is revealed in relation to the cleavage of COL2, this MMP is likely involved. The collective findings reveal that COL2 is attacked at the afore-described susceptible peptide bond at the surface of cartilage canals and, that MMP-13 cleaves it. Developmental Dynamics 238:1547-1563, 2009. (c) 2009 Wiley-Liss, Inc.
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http://dx.doi.org/10.1002/dvdy.21960DOI Listing
June 2009

The pattern recognition receptor CD36 is a chondrocyte hypertrophy marker associated with suppression of catabolic responses and promotion of repair responses to inflammatory stimuli.

J Immunol 2009 Apr;182(8):5024-31

Department of Medicine, Rheumatology Section, Veterans Affairs Health Care System/University of California, San Diego, CA 92161, USA.

Multiple inflammatory mediators in osteoarthritis (OA) cartilage, including S100/calgranulin ligands of receptor for advanced glycation end products (RAGE), promote chondrocyte hypertrophy, a differentiation state associated with matrix catabolism. In this study, we observed that RAGE knockout was not chondroprotective in instability-induced knee OA in 8-wk-old mice. Hence, we tested the hypothesis that expression of the alternative S100/calgranulin and patterning receptor CD36, identified here as a marker of growth plate chondrocyte hypertrophy, mediates chondrocyte inflammatory and differentiation responses that promote OA. In rat knee joint destabilization-induced OA, RAGE expression was initially sparse throughout cartilage but increased diffusely by 4 wk after surgery. In contrast, CD36 expression focally increased at sites of cartilage injury and colocalized with developing chondrocyte hypertrophy and aggrecan cleavage NITEGE neoepitope formation. However, CD36 transfection in normal human knee-immortalized chondrocytes (CH-8 cells) was associated with decreased capacity of S100A11 and TNF-alpha to induce chondrocyte hypertrophy and ADAMTS-4 and matrix metalloproteinase 13 expression. S100A11 lost the capacity to inhibit proteoglycans synthesis and gained the capacity to induce proteoglycan synthesis in CD36-transfected CH-8 cells. Moreover, S100A11 required the p38 MAPK pathway kinase MKK3 to induce NITEGE development in mouse articular cartilage explants. However, CH-8 cells transfected with CD36 demonstrated decreased S100A11-induced MKK3 and p38 phosphorylation. Therefore, RAGE and CD36 patterning receptor expression were linked with opposing effects on inflammatory, procatabolic responses to S100A11 and TNF-alpha in chondrocytes.
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http://dx.doi.org/10.4049/jimmunol.0803603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2698125PMC
April 2009

Hyaluronidase 3 (HYAL3) knockout mice do not display evidence of hyaluronan accumulation.

Matrix Biol 2008 Oct 14;27(8):653-60. Epub 2008 Aug 14.

Department of Biochemistry and Medical Genetics, The University of Manitoba, Winnipeg, MB, Canada.

Hyaluronidases are endoglycosidases that initiate the breakdown of hyaluronan (HA), an abundant component of the vertebrate extracellular matrix. In humans, six paralogous genes encoding hyaluronidase-like sequences have been identified on human chromosomes 3p21.3 (HYAL2-HYAL1-HYAL3) and 7q31.3 (SPAM1-HYAL4-HYALP1). Mutations in one of these genes, HYAL1, were reported in a patient with mucopolysaccharidosis (MPS) IX. Despite the broad distribution of HA, the HYAL1-deficient patient exhibited a mild phenotype, suggesting other hyaluronidase family members contribute to constitutive HA degradation. Hyal3 knockout (Hyal3-/-) mice were generated to determine if HYAL3 had a role in constitutive HA degradation. Hyal3-/- mice were viable, fertile, and exhibited no gross phenotypic changes. X-ray analysis, histological studies of joints, whole-body weights, organ weights and the serum HA levels of Hyal3-/- mice were normal. No evidence of glycosaminoglycan accumulation, including vacuolization, was identified in the Hyal3-/- tissues analyzed. Remarkably, the only difference identified in Hyal3-/- mice was a subtle change in the alveolar structure and extracellular matrix thickness in lung-tissue sections at 12-14 months-of-age. We conclude that HYAL3 does not play a major role in constitutive HA degradation.
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http://dx.doi.org/10.1016/j.matbio.2008.07.006DOI Listing
October 2008

Displacement of the occluding loop by the parasite protein, chagasin, results in efficient inhibition of human cathepsin B.

J Biol Chem 2008 Aug 30;283(33):22815-25. Epub 2008 May 30.

Faculty of Biotechnology and Food Sciences, Technical University of Lodz, 90-924 Lodz, Poland.

Cathepsin B is a papain-like cysteine protease showing both endo- and exopeptidase activity, the latter due to a unique occluding loop that restricts access to the active site cleft. To clarify the mode by which natural protein inhibitors manage to overcome this obstacle, we have analyzed the structure and function of cathepsin B in complexes with the Trypanosoma cruzi inhibitor, chagasin. Kinetic analysis revealed that substitution of His-110e, which anchors the loop in occluding position, results in 3-fold increased chagasin affinity (Ki for H110A cathepsin B, 0.35 nm) due to an improved association rate (kon, 5 x 10(5) m(-1)s(-1)). The structure of chagasin in complex with cathepsin B was solved in two crystal forms (1.8 and 2.67 angstroms resolution), demonstrating that the occluding loop is displaced to allow chagasin binding with its three loops, L4, L2, and L6, spanning the entire active site cleft. The occluding loop is differently displaced in the two structures, indicating a large range of movement and adoption of conformations forced by the inhibitor. The area of contact is slightly larger than in chagasin complexes with the endopeptidase, cathepsin L. However, residues important for high affinity to both enzymes are mainly found in the outer loops L4 and L6 of chagasin. The chagasin-cathepsin B complex provides a structural framework for modeling and design of inhibitors for cruzipain, the parasite cysteine protease and a virulence factor in Chagas disease.
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http://dx.doi.org/10.1074/jbc.M802064200DOI Listing
August 2008

Cleavage of type II collagen by cathepsin K in human osteoarthritic cartilage.

Am J Pathol 2008 Jul 29;173(1):161-9. Epub 2008 May 29.

Joint Diseases Laboratory, Shriners Hospitals for Children, 1529 Cedar Ave., Montreal, Quebec, H3G1A6, Canada.

Cathepsin K is a cysteine protease of the papain family that cleaves triple-helical type II collagen, the major structural component of the extracellular matrix of articular cartilage. In osteoarthritis (OA), the anabolic/catabolic balance of articular cartilage is disrupted with the excessive cleavage of collagen II by collagenases or matrix metalloproteinases. A polyclonal antibody against a C-terminal neoepitope (C2K) generated in triple-helical type II collagen by the proteolytic action of cathepsin K was prepared and used to develop an enzyme-linked immunosorbent assay to study the generation of this epitope and the effects of its presence in normal adult and osteoarthritic femoral condylar articular cartilage. The generation of the C2K epitope in explant culture and the effect of a specific cathepsin K inhibitor were studied. The neoepitope, which is not generated by the collagenase matrix metalloproteinase-13, increased with age in articular cartilage and was significantly elevated in osteoarthritic cartilage compared with adult nonarthritic cartilage. Moreover, in explants from three of eight OA patients, the generation of the neoepitope in culture was significantly reduced by a specific, nontoxic inhibitor of cathepsin K. These data suggest that cathepsin K is involved in the cleavage of type II collagen in human articular cartilage in certain OA patients and that it may play a role in both OA pathophysiology and the aging process.
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http://dx.doi.org/10.2353/ajpath.2008.070494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2438294PMC
July 2008

A mouse model of human mucopolysaccharidosis IX exhibits osteoarthritis.

Hum Mol Genet 2008 Jul 15;17(13):1904-15. Epub 2008 Mar 15.

Department of Biochemistry and Medical Genetics, University of Manitoba, 770 Bannatyne Avenue, Winnipeg, MB, Canada R3E 0W3.

Hyaluronidases are endoglycosidases that hydrolyze hyaluronan (HA), an abundant component of the extracellular matrix of vertebrate connective tissues. Six human hyaluronidase-related genes have been identified to date. Mutations in one of these genes cause a deficiency of hyaluronidase 1 (HYAL1) resulting in a lysosomal storage disorder, mucopolysaccharidosis (MPS) IX. We have characterized a mouse model of MPS IX and compared its phenotype with the human disease. The targeted Hyal1 allele in this model had a neomycin resistance cassette in exon 2 that replaced 753 bp of the coding region containing the predicted enzyme active site. As a result, Hyal1(-/-) animals had no detectable wild-type Hyal1 transcript, protein or serum activity. Hyal1 null animals were viable, fertile and showed no gross abnormalities at 1 year and 8 months of age. Histological studies of the knee joint showed a loss of proteoglycans occurring as early as 3 months that progressed with age. An increased number of chondrocytes displaying intense pericellular and/or cytoplasmic HA staining were detected in the epiphyseal and articular cartilage of null mice, demonstrating an accumulation of HA. Elevations of HA were not detected in the serum or non-skeletal tissues, indicating that osteoarthritis is the key disease feature in a Hyal1 deficiency. Hyal3 expression was elevated in Hyal1 null mice, suggesting that Hyal3 may compensate in HA degradation in non-skeletal tissues. Overall, the murine MPS IX model displays the key features of the human disease.
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http://dx.doi.org/10.1093/hmg/ddn088DOI Listing
July 2008

Measurement of glycosaminoglycan release from cartilage explants.

Methods Mol Med 2007 ;135:201-9

Quantitation of glycosaminoglycans (GAGs) in the form of aggrecan fragments released from cartilage in culture is a simple way to determine the efficacy of different cytokines alone or in combination in simulating cartilage catabolism. Two approaches for GAG assay are described, with special attention being paid to the advantages and limitations of each method.
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http://dx.doi.org/10.1007/978-1-59745-401-8_12DOI Listing
December 2007

Crystal structure of the parasite protease inhibitor chagasin in complex with a host target cysteine protease.

J Mol Biol 2007 Aug 10;371(1):137-53. Epub 2007 May 10.

Department of Laboratory Medicine, Division of Clinical Chemistry and Pharmacology, Lund University, University Hospital, SE-221 85 Lund, Sweden.

Chagasin is a protein produced by Trypanosoma cruzi, the parasite that causes Chagas' disease. This small protein belongs to a recently defined family of cysteine protease inhibitors. Although resembling well-known inhibitors like the cystatins in size (110 amino acid residues) and function (they all inhibit papain-like (C1 family) proteases), it has a unique amino acid sequence and structure. We have crystallized and solved the structure of chagasin in complex with the host cysteine protease, cathepsin L, at 1.75 A resolution. An inhibitory wedge composed of three loops (L2, L4, and L6) forms a number of contacts responsible for high-affinity binding (K(i), 39 pM) to the enzyme. All three loops interact with the catalytic groove, with the central loop L2 inserted directly into the catalytic center. Loops L4 and L6 embrace the enzyme molecule from both sides and exhibit distinctly different patterns of protein-protein recognition. Comparison with a 1.7 A structure of uncomplexed chagasin, also determined in this study, demonstrates that a conformational change of the first binding loop (L4) allows extended binding to the non-primed substrate pockets of the enzyme active site cleft, thereby providing a substantial part of the inhibitory surface. The mode of chagasin binding is generally similar, albeit distinctly different in detail, when compared to those displayed by cystatins and the cysteine protease inhibitory p41 fragment of the invariant chain. The chagasin-cathepsin L complex structure provides details of how the parasite protein inhibits a host enzyme of possible importance in host defense. The high level of structural and functional similarity between cathepsin L and the T. cruzi enzyme cruzipain gives clues to how the cysteine protease activity of the parasite can be targeted. This information will aid in the development of synthetic inhibitors for use as potential drugs for the treatment of Chagas disease.
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http://dx.doi.org/10.1016/j.jmb.2007.05.005DOI Listing
August 2007

Increased collagen and aggrecan degradation with age in the joints of Timp3(-/-) mice.

Arthritis Rheum 2007 Mar;56(3):905-9

Shriners Hospital for Children and McGill University, Montreal, Quebec, Canada.

Objective: To investigate the in vivo effect of an imbalance between metalloproteinases and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), in mouse articular cartilage.

Methods: Hind joints of Timp3(-/-) and wild-type mice were examined by routine staining and by immunohistochemical analysis using antibodies specific for type X collagen and for the neoepitopes produced on proteolytic cleavage of aggrecan (... VDIPEN and ... NVTEGE) and type II collagen. The neoepitope generated on cleavage of type II collagen by collagenases was quantitated in sera by enzyme-linked immunosorbent assay.

Results: Articular cartilage from Timp3-knockout animals (ages > or =6 months) showed reduced Safranin O staining and an increase in ...VDIPEN content compared with cartilage from heterozygous and wild-type animals. There was also a slight increase in ... NVTEGE content in articular cartilage and menisci of Timp3(-/-) animals. Chondrocytes showed strong pericellular staining for type II collagen cleavage neoepitopes, particularly in the superficial layer, in knockout mice. Also, there was more type X collagen expression in the superficial zone of articular cartilage, especially around clusters of proliferating chondrocytes, in the knockout mice. More type II collagen cleavage product was found in the serum of Timp3(-/-) mice compared with wild-type animals. This increase was significant in 15-month-old animals.

Conclusion: These results indicate that TIMP-3 deficiency results in mild cartilage degradation similar to changes seen in patients with osteoarthritis, suggesting that an imbalance between metalloproteinases and TIMP-3 may play a pathophysiologic role in the development of this disease.
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http://dx.doi.org/10.1002/art.22427DOI Listing
March 2007

Quantitative continuous assay for hyaluronan synthase.

Anal Biochem 2007 Feb 27;361(2):218-25. Epub 2006 Nov 27.

Joint Diseases Laboratory, Shriners Hospital for Children, Montreal, Que., Canada H3G 1A6.

A rapid, continuous, and convenient three-enzyme coupled UV absorption assay was developed to quantitate the glucuronic acid and N-acetylglucosamine transferase activities of hyaluronan synthase from Pasteurella multocida (PmHAS). Activity was measured by coupling the UDP produced from the PmHAS-catalyzed transfer of UDP-GlcNAc and UDP-GlcUA to a hyaluronic acid tetrasaccharide primer with the oxidation of NADH. Using a fluorescently labeled primer, the products were characterized by gel electrophoresis. Our results show that a truncated soluble form of recombinant PmHAS (residues 1-703) can catalyze the glycosyl transfers in a time- and concentration-dependent manner. The assay can be used to determine kinetic parameters, inhibition constants, and mechanistic aspects of this enzyme. In addition, it can be used to quantify PmHAS during purification of the enzyme from culture media.
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http://dx.doi.org/10.1016/j.ab.2006.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4114249PMC
February 2007

The structure and degradation of aggrecan in human intervertebral disc.

Eur Spine J 2006 Aug 31;15 Suppl 3:S326-32. Epub 2006 May 31.

Genetics Unit, Shriners Hospital for Children, Montreal, QC, Canada.

The ability of the intervertebral disc to resist compression is dependent on its high proteoglycan concentration. The disc proteoglycans are classified as aggregating or non-aggregating depending on their ability to interact with hyaluronan. The majority of the aggregating proteoglycans are derived from aggrecan, though their glycosaminoglycan substitution pattern has not been determined. In contrast, the origin of the non-aggregating proteoglycans is unclear, though it has been postulated that they are derived from aggrecan by proteolysis. The present work demonstrates that keratan sulfate (KS) in the glycosaminoglycan-binding region of disc aggrecan is confined to the KS-rich domain of the core protein and is not present in association with chondroitin sulfate (CS) in the CS1 and CS2 domains. It also shows that the non-aggregating disc proteoglycans are derived from aggrecan, with the large molecules possessing both the KS-rich and CS1 domains and the smaller molecules being generated from either the KS-rich or CS2 domain. The origin and spectrum of disc proteoglycan heterogeneity is the same in both the annulus fibrosus and nucleus pulposus.
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http://dx.doi.org/10.1007/s00586-006-0127-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2335376PMC
August 2006

Protease analysis by neoepitope approach reveals the activation of MMP-9 is achieved proteolytically in a test tissue cartilage model involved in bone formation.

J Histochem Cytochem 2006 Sep 18;54(9):965-80. Epub 2006 May 18.

Electron Microscopy Unit, Joint Diseases Laboratory, Shriners Hospital for Children, and Division of Surgical Research, Department of Surgery, McGill University, Montreal, Quebec, H3G 1A6, Canada.

A principle of regulation of matrix metalloproteinase (MMP) activity has been introduced as the cysteine-switch mechanism of activation (Springman et al. 1990). According to this mechanism, a critical Cys residue found in the auto-inhibitory propeptide domain of latent proenzyme is important to determine whether or not activation is turned on or off. The mechanism further allows for multiple modes of activation. To determine whether or not activation is accomplished proteolytically within a rat test cartilage model, protease analysis by the neoepitope approach, which relies upon a set of antibodies, was applied. One is used to identify the MMP-9 proenzyme bearing the critical cysteine residue, the other to identify any enzyme present bearing a new NH2-terminus 89FQTFD. This is indicative of MMP-9 lacking the cysteine switch. The antibody set has been applied to frozen tissue sections and analyzed by light and electron microscopic methods. Results reveal that activation of the MMP-9 protease involves limited proteolysis resulting in propeptide domain release. Here we report the observed changes of protease form to indigenous cells and extracellular matrix, thereby making it possible to uncover the features of MMP-9 activation within a specified set of tissue circumstances where a cartilage model is transformed into definitive bone. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.
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http://dx.doi.org/10.1369/jhc.5A6789.2006DOI Listing
September 2006

The cysteine-rich domain of the secreted proprotein convertases PC5A and PACE4 functions as a cell surface anchor and interacts with tissue inhibitors of metalloproteinases.

Mol Biol Cell 2005 Nov 31;16(11):5215-26. Epub 2005 Aug 31.

Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Quebec H2W 1R7, Canada.

The proprotein convertases PC5, PACE4 and furin contain a C-terminal cysteine-rich domain (CRD) of unknown function. We demonstrate that the CRD confers to PC5A and PACE4 properties to bind tissue inhibitors of metalloproteinases (TIMPs) and the cell surface. Confocal microscopy and biochemical analyses revealed that the CRD is essential for cell surface tethering of PC5A and PACE4 and that it colocalizes and coimmunoprecipitates with the full-length and C-terminal domain of TIMP-2. Surface-bound PC5A in TIMP-2 null fibroblasts was only observed upon coexpression with TIMP-2. In COS-1 cells, plasma membrane-associated PC5A can be displaced by heparin, suramin, or heparinases I and III and by competition with excess exogenous TIMP-2. Furthermore, PC5A and TIMP-2 are shown to be colocalized over the surface of enterocytes in the mouse duodenum and jejunum, as well as in liver sinusoids. In conclusion, the CRD of PC5A and PACE4 functions as a cell surface anchor favoring the processing of their cognate surface-anchored substrates, including endothelial lipase.
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http://dx.doi.org/10.1091/mbc.e05-06-0504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1266420PMC
November 2005

The concentration, gene expression, and spatial distribution of aggrecan in canine articular cartilage, meniscus, and anterior and posterior cruciate ligaments: a new molecular distinction between hyaline cartilage and fibrocartilage in the knee joint.

Connect Tissue Res 2005 ;46(2):83-91

Department of Biomedical Engineering, Lerner Research Institute and Orthopaedic Research Center, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.

The concentration, spatial distribution, and gene expression of aggrecan in meniscus, articular cartilage, and the anterior and posterior cruciate ligaments (ACL and PCL) was determined in the knee joints of five mature dogs. An anti-serum against peptide sequences specific to the G1 domain of aggrecan was employed in competitive-inhibition ELISA of guanidine HCl extracts and immunofluorescence microscopy. Gene expression was determined by Taqman real-time PCR. The concentration of aggrecan in articular cartilage (240.1 +/- 32 nMol/g dry weight) was higher than that in meniscus (medial meniscus: 33.4 +/- 4.3 nMol/g) and ligaments (ACL: 6.8 +/- 0.9 nMol/g). Aggrecan was more concentrated in the inner than the outer zone of the meniscus. Aggrecan in meniscus showed an organized, spatial network, in contrast to its diffuse distribution in articular cartilage. Thus, differences in the concentration, gene expression, and spatial distribution of aggrecan constitute another molecular distinction between hyaline cartilage and fibrocartilage of the knee.
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http://dx.doi.org/10.1080/03008200590954113DOI Listing
September 2005
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