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Osteoarthritis Cartilage. Author manuscript; available in PMC 2011 Mar 1.
Published in final edited form as:
Osteoarthritis Cartilage. 2010 Mar; 18(3): 455–463.
Published online 2009 Nov 4. doi:  10.1016/j.joca.2009.10.014
PMCID: PMC2826559

10 mM glucosamine prevents activation of proADAMTS5 (aggrecanase-2) in transfected cells by interference with post-translational modification of furin



Glucosamine has been previously shown to suppress cartilage aggrecan catabolism in explant cultures. We determined the effect of glucosamine on ADAMTS5, a major aggrecanase in osteoarthritis, and investigated a potential mechanism underlying the observed effects.


HEK293F and CHO-K1 cells transiently transfected with ADAMTS5 cDNA were treated with glucosamine or the related hexosamine mannosamine. Glucosamine effects on FURIN transcription were determined by quantitative RT-PCR. Effects on furin-mediated processing of ADAMTS5 zymogen, and aggrecan processing by glucosamine-treated cells, were determined by western blotting. Post-translational modification of furin and N-glycan deficient furin mutants generated by site-directed mutagenesis was analyzed by western blotting, and the mutants were evaluated for their ADAMTS5 processing ability in furin-deficient CHO-RPE.40 cells.


10 mM glucosamine and 5–10 mM mannosamine reduced excision of the ADAMTS5 propeptide, indicating interference with the propeptide excision mechanism, although mannosamine compromised cell viability at these doses. Although glucosamine had no effect on furin mRNA levels, western blot of furin from glucosamine-treated cells suggested altered post-translational modification. Glucosamine treatment led to decreased glycosylation of cellular furin, with reduced furin autoactivation as the consequence. Recombinant furin treated with peptide N-glycanase F had reduced activity against a synthetic peptide substrate. Indeed, site-directed mutagenesis of two furin N-glycosylation sites, Asn387 and Asn440, abrogated furin activation and this mutant was unable to rescue ADAMTS5 processing in furin-deficient cells.


10 mM glucosamine reduces excision of the ADAMTS5 propeptide via interference with post-translational modification of furin and leads to reduced aggrecanase activity of ADAMTS5.

Keywords: Aggrecanase, glucosamine, ADAMTS, furin, catabolism


Osteoarthritis (OA) is a common disorder characterized by loss of articular cartilage as a result of degenerative changes in the joint. An early phenomenon in cartilage degradation is proteolytic loss of aggrecan, which forms giant aggregates with hyaluronan in the extracellular matrix (ECM) [1]. These aggregates bind water, and are constrained by a collagen network, which gives cartilage its compressibility. Aggrecan proteolysis diminishes cartilage compressibility and exposes collagen and other cartilage ECM proteins to degradation, thereby setting in motion a vicious cycle of cartilage breakdown [2]. Two secreted metalloproteases, ADAMTS4 and ADAMTS5, referred to as aggrecanases, cleave aggrecan efficiently and are implicated as key mediators of OA [3, 4]. Therefore, inhibition of these proteases, such as by endogenous inhibitors, medicinal compounds, or biosynthetic interference, is of significant therapeutic interest [5]. ADAMTS5, also known as aggrecanase-2, has attracted considerable interest as a target in OA [5, 6] since mice with a targeted deletion of Adamts5 are resistant to both immune and mechanically induced arthritis [7, 8].

ADAMTS5 is synthesized as a zymogen (proADAMTS5) which undergoes proteolytic excision of its propeptide by proprotein convertases (PCs) such as furin and PACE4 [9, 10]. ADAMTS4 and ADAMTS5 each require propeptide excision for proteolytic activity, but they are activated somewhat differently. ADAMTS4 is processed intracellularly [11], whereas ADAMTS5 is processed extracellularly by furin and/or other PCs. ADAMTS4 and ADAMTS5 zymogens may also be deposited in cartilage ECM, where they are activated by secreted PCs such as PACE4 [10]. Both furin and PACE4, which activate ADAMTS5 efficiently, cleave the consensus cleavage site, RRRR261↓, which is present at the junction of the ADAMTS5 propeptide and catalytic domain [9, 10, 12].

The hexosamines glucosamine and mannosamine were previously shown to suppress aggrecan catabolism in cartilage explant cultures [1319]. Both hexosamines can interfere with N-glycosylation [20, 21], and mannosamine is a recognized inhibitor of glycosylphospatidyl-inositol (GPI) anchor formation [1419]. The effects of hexosamines on ADAMTS4 biosynthesis, but not on ADAMTS5, were previously investigated at the molecular level. C-terminal processing of furin-activated ADAMTS4 by a GPI-anchored metalloprotease located at the cell surface was inhibited by mannosamine [15, 22]. Treatment of cells with hexosamines also led to a prevalence of unprocessed ADAMTS4 zymogen [15]. ADAMTS4 lacks N-linked glycans [4], so the observed effects could not be attributed to inhibition of its N-glycosylation by hexosamines. These published observations led us to examine the effects of glucosamine on ADAMTS5 activity, and to elucidate the molecular mechanisms of the observed effects. Unlike ADAMTS4, ADAMTS5 is N-glycosylated and does not bind to the cell-surface, but has been shown to reside in the ECM [23, 24]. In cultured cells, which make little ECM, such as HEK293F cells, ADAMTS5 is present in the conditioned medium [9]. Here, we show that hexosamine treatment of cells interferes with the activation of ADAMTS5 through an indirect mechanism involving the loss of furin activity. These findings provide one possible explanation for previously observed suppressive effects of glucosamine on cartilage catabolism and are thus relevant to OA biochemistry.

Materials and Methods


HEK293F cells and CHO-K1 cells (ATCC, Manassus, VA) were cultured in Dulbecco’s Modified Eagles Medium (DMEM, high glucose) supplemented with 10 % FBS and antibiotics. Furin-deficient CHO-RPE.40 cells [25] were cultured in Ham’s F12 medium (high glucose) supplemented with 10 % FBS and antibiotics.


Constructs for expression of full-length human ADAMTS5 and the propeptide and catalytic domain of human ADAMTS5 (ADAMTS5 Pro-Cat) were previously described [9] (Fig. 1). Asn residues within two of three consensus N-linked glycosylation sites in furin (GenBank accession no. NP_002560) were mutated singly (Asn387, Asn440), or in combination (Asn387+440), by replacement with Gln using site-directed mutagenesis (Quikchange site-directed mutagenesis kit, Stratagene, La Jolla, CA). The mutations were introduced into full-length untagged human furin and a furin construct with an N-terminal FLAG tag introduced immediately downstream of the furin autoactivation site [26].

Figure 1
ADAMTS5 constructs used in the analysis of glucosamine effects

Expression plasmids were transiently transfected into HEK293F, CHO-K1 and CHO RPE.40 cells using FUGENE6 (Roche Diagnostics, Indianapolis, IN). HEK293F and CHO-K1 cells were treated with glucosamine-HCl (neutralized to pH 7.4 before use) or mannosamine (Sigma-Aldrich, St. Louis, MO). Before treatment, cells were transferred to serum-free DMEM containing 5 mM glucose for 3–5 h [13]. Fresh medium containing each compound in a range of concentrations was added to cells. Conditioned medium was collected, and cells were harvested in phosphate buffered saline after 24 h or 48 h followed by lysis in 1% Triton X-100, 10 mM Tris.HCl, pH 7.6 containing complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). In some experiments, HEK293F and CHO RPE.40 cells were transfected (or co-transfected) with furin (either wildtype or Asn→Gln mutants) and/or ADAMTS5 Pro-Cat plasmid. As a control, empty vector used for furin cloning (pCI-Neo, Promega, Madison, WI) was co-transfected with ADAMTS5 Pro-Cat in CHO-RPE.40 cells. Serum-free media and cell lysates were collected after 48 h and processed as described above.


HEK293F cells were treated with glucosamine and harvested after 48 h as described above. Total RNA was extracted using QIAGEN complete RNA isolation kit (QIAGEN, Valencia, CA). RNA concentration and purity were determined with a NanoDrop (ND-100) spectrophotometer (Nanodrop Technologies, Wilmington, DE). All RNA samples used had an A260/280 ratio of >1.8. Two μg of total RNA per condition was reverse transcribed to cDNA in a total volume of 20 μl using the Superscript III kit (Invitrogen, Carlsbad, CA). For quantitative PCR, the FURIN primers 5′-CAGCGGTGGCCAACAGTGTG-3′ (forward) 5′-GCGGGCGGTGAGGCGACA-3′ (reverse) and 18s ribosomal RNA primers, 5′-GGGAGGTAGTGACGAAAAATAACAAT-3′ (forward), 5′-TTGCCCTCCAATGGATCCT-3′(reverse) were used. SYBR-Green (Applied Biosystems, Foster City, CA) incorporation was detected using an iCycler (Bio-Rad. Hercules, CA) and relative levels of FURIN mRNA were quantitated using the ΔΔct method as per manufacturer’s protocol.


For western blots, conditioned medium and cell lysate were electrophoresed by reducing SDS-PAGE in 6% or 7.5% gels prior to electroblotting to polyvinylidene fluoride membrane (Millipore, Billerica, MA). Peptide N-glycosidase F (PNGase F, New England Biolabs, Ipswich, MA) treatment was carried out as per manufacturer’s instructions following reduction of the sample. ADAMTS5 was detected using rabbit polyclonal antibody RP-2 (recognizing the propeptide) (Triple Point Biologics, Forest Grove, OR) [27] or anti-myc mouse monoclonal antibody 9E10 (Sigma-Aldrich, St. Louis, MO). Anti-GAPDH mouse monoclonal antibody (Chemicon, Temecula, CA) was used to detect intracellular and extracellular GAPDH to ensure equivalent protein loading. Anti-furin mouse monoclonal antibody MON-152 recognizing an epitope in the cysteine-rich domain (Alexis Biochemicals, San Diego, CA) was used for detection of endogenous and untagged furin, whereas mouse anti-FLAG M1 or M2 monoclonal antibody and rabbit anti-FLAG polyclonal antibody were used for detection of FLAG-tagged furin (FLAG antibodies from Sigma-Aldrich, St. Louis, MO). Anti-FLAG M2 antibody recognizes the FLAG tag at any position within the protein, whereas anti-FLAG M1 preferentially recognizes a FLAG tag at the free N-terminus. Antibody binding was detected by enhanced chemiluminescence (ECL, GE Healthcare, Piscataway, NJ). For detection of endogenous furin, ECL-Plus (GE Healthcare, Piscataway, NJ) was utilized. Intensity of relevant bands was quantitated using IMAGE J software (NIH, Bethesda, MD). A type 1 (paired), 2-tailed student’s t-test was used to determine whether the normalized data obtained from the ImageJ software was statistically significant (P= <0.05) between the control and test group, Gaussian distribution was assumed.

ADAMTS5 cleavage of aggrecan at the Glu373-Ala374 peptide bond was determined using a modification of a previously described experimental procedure [9]. HEK293F cells were transfected with full-length ADAMTS5 followed by the addition of serum-free medium with or without 10 mM glucosamine. Conditioned medium (CM) was collected after 48 h. Fifty μl of CM from control (untransfected) or from each respective ADAMTS5 transfection was incubated with 4 μg bovine aggrecan (Sigma–Aldrich) for 16 h at37 °C in the presence of an EDTA-free protease inhibitor cocktail (Roche). Each reaction was heat-inactivated (5 min, 100 °C), cooled on ice, and aggrecan was deglycosylated with protease-free chondroitinase ABC, keratanase I and keratanase II (Seikagaku, Tokyo, Japan) at 37 °C for 2 h. Western blotting was performed using the anti-NITEGE aggrecan neo-epitope antibody that specifically detects cleavage at the Glu373-Ala374 peptide bond (kindly provided by Dr. John Sandy, Rush University Medical Center). ADAMTS5 zymogen, mature ADAMTS5 and ADAMTS5 propeptide in the CM were detected by western blot. Anti-GAPDH was used as a control to demonstrate comparable cell numbers.


Recombinant human furin (rhFurin, purity >95%) was purchased from R&D systems (Minneapolis, MN). Fifty or 100 ng of rhFurin per reaction was treated with PNGaseF (New England Biolabs) or with heat-inactivated PNGaseF (100 °C, 5 min) as a control in HEPES buffer (100 mM HEPES, 0.5% Triton X-100, 1 mM CaCl2, 1 mM β-mercaptoethanol, pH7.5) for 2 h at 37 °C. Substrate cleavage was measured by the liberation of AMC from Boc-RVRR-AMC (final concentration 100 μM, Biomol, Plymouth Meeting, PA) in 100 μl HEPES buffer, pH 7.5. Fluorescence was measured every minute (total 90 min) on a Victor 2 1420 Multilabel Counter fluorescent plate reader (Perkin Elmer, Waltham, MA) with excitation/emission wavelength of 355/460 nm respectively. Each assay was run in triplicate in two separate assays in 96-well plates. The extinction co-efficient (ε) of AMC was determined to be 17.3 μm−1cm−1 using an evenly spaced standard curve (range 40 – 400 μm). Specific activity was determined for each individual reaction using the following formula: μm/min = ([Rate (OD/min)/ε] × 1e-4l). Statistical analysis was performed using a Student t-test on the raw data generated for control (glycosylated) and test (deglycoslated) furin activity. The data are presented as the mean specific activity of 3 individual rates per assay +/− standard deviation.



To investigate a possible effect of glucosamine on ADAMTS5, myc-tagged full-length ADAMTS5 (Fig. 1) was transiently transfected in HEK293F cells and the molecular species present in the conditioned medium were evaluated by western blotting with anti-myc antibody and anti-propeptide antibody. The medium of untreated cells showed the ADAMTS5 zymogen (120 kDa) and mature ADAMTS5 (85 kDa) using anti-myc, whereas the zymogen and cleaved 25 kDa propeptide were recognized by anti-RP2 (Fig. 2A). The relatively weak reactivity of unprocessed ADAMTS5 with anti-RP2, compared to anti-myc, likely reflects the stronger affinity of the myc-antibody. The relatively low amount of the mature ADAMTS5 in medium possibly reflects the previously reported observation that heparin specifically enhances the detection of mature ADAMTS5 in medium of these cells. Thus mature ADAMTS5 may be sequestered in the extracellular matrix by ionic interactions. Mature ADAMTS5 detected by anti- myc was decreased in the presence of 5mM and 10 mM glucosamine with a corresponding increase in the intact zymogen (Fig. 2A, top panel). Reduction of ADAMTS5 activation, as represented by a decrease in proteolytically cleaved propeptide (detected by RP2, Fig. 2A, second panel from bottom) in conditioned medium, was observed upon treatment with 5 and 10 mM glucosamine in both HEK293F cells (Fig. 2A, B) and CHO-K1 cells (data not shown).

Figure 2Figure 2
ADAMTS5 activation is inhibited by glucosamine treatment

Medium from ADAMTS5-expressing cells treated with 10 mM glucosamine contained significantly less proteolytic activity when cleavage of the Glu373-Ala374 bond of aggrecan core protein was compared to untreated cells, consistent with reduced proADAMTS5 activation (Fig. 2C). This suggested that aggrecan proteolysis by ADAMTS5 was profoundly affected by 10 mM glucosamine due to impaired ADAMTS5 activation. Similarly, we found that constructs of ADAMTS9 and ADAMTS20 expressed in HEK293F cells also had reduced propeptide processing in the presence of 10 mM glucosamine (data not shown). We concluded that reduction of ADAMTS5-dependent aggrecan processing was probably not a direct effect of glucosamine on ADAMTS5, but an indirect effect on the processing mechanism.

Treatment of transfected HEK293F cells with 5 mM or 10 mM mannosamine also resulted in decreased removal of the ADAMTS5 propeptide (Fig. 3A). Because high levels of hexosamines may have deleterious effects on cells, we analyzed cell viability at the effective glucosamine and mannosamine concentrations tested, as well as 25 mM glucosamine. There was decreased cell viability in the presence of 25 mM glucosamine, as well as 5 mM and 10 mM mannosamine (Fig. 3B). Therefore, we primarily investigated the effects of 10 mM glucosamine, since this did not affect cell viability.

Figure 3Figure 3
Effect of mannosamine on ADAMTS5 propeptide processing, and cell viability after glucosamine and mannosamine treatment with various doses


Previous work has established the role of furin in ADAMTS zymogen processing, including ADAMTS5 [9, 24, 28, 29]. Glucosamine is reported to affect transcription of many genes [30, 31], prompting us to examine whether it affected furin transcription. Quantitative RT-PCR demonstrated no significant change in furin mRNA in 10 mM glucosamine-treated HEK293F cells (Fig. 4A). Next, we investigated the effect of glucosamine on endogenous cellular furin by western blot analysis. Furin is detected as a doublet of the zymogen (90 kDa) and activated enzyme (85 kDa), in untreated HEK293F cells (Fig. 4B). In the presence of 5mM and 10 mM glucosamine, there was an increased intensity of a furin species apparently co-migrating with active furin in untreated cells (Fig. 4B). However, since the activity of furin was clearly reduced in the presence of 10 mM glucosamine (Fig. 2), we concluded that this altered electrophoretic migration likely represented reduced furin glycosylation rather than increased profurin conversion to active furin, consistent with a role for glucosamine in inhibition of N-glycosylation [20]. The reduced furin activity in the presence of glucosamine suggested that 85 kDa furin species not only lacked glycosylation, but also failed to be processed in the presence of 10 mM glucosamine. The enhanced furin immunoreactivity seen (Fig. 4B) could also result from blockade of furin shedding from the cell surface but could not be resolved by analysis of the conditioned medium of cells owing to the relatively low levels present in medium. These data strongly suggested that glucosamine impaired furin function via an effect on post-translational modification of furin, such as altered glycosylation, decreased conversion to furin, decreased shedding or a combination of these.

Figure 4
Glucosamine affects post-translational modification of furin


To resolve these various possibilities, we first examined whether mature furin was dependent on N-glycosylation for its activity. Recombinant, purified, N-terminally His-tagged human furin was treated with PNGaseF to eliminate N-linked sugars. Furin activity towards the fluorogenic substrate Boc-RVRR-AMC was significantly impaired after treatment with PNGaseF in two independent assays (Fig. 5A, B). Western blotting confirmed complete deglycosylation of the PNGaseF treated furin used in these assays (Fig. 5C). However, since the activity of mature furin was only partially abolished by enzymatic deglycosylation, we investigated the effect of glucosamine on cellular furin.

Figure 5
Deglycosylation of purified recombinant furin reduces its activity

To better resolve the effect of glucosamine on profurin conversion and shedding, we used plasmids for expression of wild-type profurin or a profurin construct containing a FLAG tag downstream of the autocatalytic processing site in HEK293F cells (Fig. 6A). Indeed, studies examining the fate of furin have typically utilized such constructs since endogenous furin is poorly detected [32, 33]. Since profurin conversion to the active form is an intrinsic, autocatalytic process [34], over-expression of these constructs by transient transfection is valid for extrapolation of any observed changes in activation status to endogenous furin. To better visualize the active form of furin, we used the anti-FLAG M1 antibody, which is reported to have increased reactivity to free N-terminal FLAG epitopes, since upon autoactivation, the tagged construct will have a free N-terminal FLAG tag (Fig. 6A). N-terminal FLAG-tagged furin, or respective mutants of the N-linked carbohydrate attachment sites (Asn387Gln, Asn440Gln and Asn387+440Gln) were transfected into HEK293F cells. Cells transfected with FLAG-tagged furin were also treated with or without 50μM dec-RVKR-CMK, a furin inhibitor, to inhibit autocatalytic activation. Anti-FLAG M1 antibody detected furin species at ~90 kDa (profurin) and ~85 kDa (zymogen and mature furin respectively, Fig. 6B) in untreated cells. Cells treated with dec-RVKR-CMK contained only furin zymogen (Fig. 6B). Asn387Gln and Asn440Gln mutants showed an expected increase in electrophorectic mobility of both furin species, but the relative position of the zymogen with respect to the active form in each of these mutants was similar to the wild-type furin (Fig. 6B). Active furin was undetectable in the Asn387+440Gln mutant (Fig. 6B), suggesting that lack of glycosylation at these two sites impaired furin autoactivation. The migration of Asn387+440Gln profurin at a size intermediate between wild-type profurin and active furin therefore appears to reflect the net effect of reduced glycosylation and impaired autoactivation, suggesting that the 85 kDa band noted in cells treated with 10 mM glucosamine (Fig. 4B) could be profurin deficient in N-glycosylation, and having reduced autoactivation. All transfected furin constructs were shed from cells, and their reduced molecular mass relative to that observed for cellular furin (Fig. 6C) was consistent with C-terminal processing of the membrane-anchored enzyme [26].

Figure 6Figure 6Figure 6
Mutation of the N-glycosylation sites of furin inhibits its autoactivation

Since ablation of N-linkage sites and 10 mM glucosamine each led to enhanced electrophoretic mobility of furin, we considered whether glucosamine may hinder furin autoactivation by interference with its glycosylation. Accordingly, we compared the effect of glucosamine on FLAG-tagged furin directly alongside the N-glycosylation mutants. Anti-FLAG M1 antibody showed enhanced mobility of furin species originating from the FLAG-tagged furin in the medium of cells treated with 10 mM glucosamine, where two bands were detected, the lower band migrated similarly to the Asn387+440 Gln furin mutant (Fig. 6D). The two molecular species seen in the medium of 10 mM glucosamine-treated cells were spaced more closely together than the two species present in medium of untreated cells (Fig. 6D). This is suggestive of both species originating from the zymogen with impaired activation and impaired glycosylation. To resolve this, we enzymatically deglycosylated the conditioned medium obtained from cells transfected with wild-type furin, furin mutants, and 10 mM glucosamine treated cells expressing wild-type furin. Following enzymatic deglycosylation, furin from the medium of 10 mM glucosamine treated cells co-migrated with the deglycosylated wild-type profurin and mutant profurin (Fig. 6E). This result establishes that treatment with 10 mM glucosamine severely compromises furin glycosylation. Since N-glycosylation is a co-translational event and precedes furin autoactivation in the trans-Golgi, this would argue that the observed lack of furin activation upon 10 mM glucosamine treatment could be secondary to impaired glycosylation of furin.

To determine whether elimination of one or more furin N-linked glycosylation sites did indeed reduce furin activity, CHO-RPE.40 cells that are deficient in furin activity were co-transfected with WT-furin (to eliminate potential functional interference by a FLAG-tag), Asn387Gln-furin, Asn440Gln-furin, or Asn387+440Gln furin mutants, together with ADAMTS5 Pro-Cat. A low level of baseline processing of ADAMTS5 Pro-Cat was seen in CHO-RPE.40 cells (presumably mediated by other convertases), but was markedly increased by co-expression of wild-type furin (Fig. 7A). In contrast, Asn440Gln furin and Asn387+440Gln furin, but not the Asn387Gln-furin mutant, showed statistically significant loss of ADAMTS5 Pro-Cat processing (Fig. 7A, B). The complete loss of activity of Asn387+440Gln furin in regard to propeptide excision (Fig. 7A, B) was consistent with the observed lack of autocatalytic activation in this mutant (Fig. 6B).

Figure 7
Glycosylation-deficient furin mutants cannot process ADAMTS5 zymogen in CHO.RPE40 cells


Glucosamine is widely utilized as a presumed anabolic agent, based on it being a precursor of cartilage glycosaminoglycans [35]. Although patients often report symptomatic benefit from glucosamine or a combination of glucosamine and chondroitin-sulfate, prospective randomized clinical trials have not consistently supported a beneficial effect [36]. On the other hand, in vitro studies demonstrated suppression of the catabolic response of cartilage explants and chondrocytes to retinoic acid and interleukin-1 in the presence of glucosamine at similar concentrations used here [13, 16, 18, 19]. Inhibition of aggrecan catabolism in cartilage explants in the presence of 5–10 mM glucosamine was reversible after removal of glucosamine [16]. Glucosamine lacks a direct inhibitory effect on soluble aggrecanase activity, and was only anti-catabolic in explant cultures undergoing a catabolic stimulus [16]. Taken together, the previously published work strongly suggested an effect of glucosamine on post-translational modification of aggrecanases. Indeed, molecular analysis of ADAMTS5 in this study demonstrates that 10 mM glucosamine impairs its activation by furin. Glucosamine is an inhibitor of N-linked glycosylation [20], yet no consistent effect was seen on electrophoretic mobility of ADAMTS5. We found that glucosamine led to altered furin migration on gels, as well as reduced activity upon elimination of N-linked carbohydrate in mature furin. Altered in-gel furin migration and concomitant loss of processing activity upon treatment of cells with 10 mM glucosamine leads us to propose that impairment of furin glycosylation compromises its conversion to active furin, which secondarily leads to failed activation of ADAMTS zymogen.

The mechanisms uncovered in this study may provide insights on several previously published observations regarding the effect of glucosamine and mannosamine on aggrecan catabolism in cartilage [13, 16, 18, 19]. Suppression of aggrecan catabolism by glucosamine, which we confirmed here, could be explained by the suppression of activation of both major aggrecanases in cartilage explants by glucosamine, since removal of the ADAMTS4 and ADAMTS5 propeptides is essential for their catalytic activity [10, 27]. The lack of N-glycosylation sites in ADAMTS4 argues that decreased ADAMTS4 [15] or ADAMTS5 activation in the presence of glucosamine is not a consequence of altered glycosylation of these ADAMTS proteases, and indeed, there was no clear effect of glucosamine on migration of ADAMTS5, which is N-glycosylated. It was previously demonstrated that pretreatment of cartilage explants with glucosamine did not confer a protective effect on the subsequent exposure to IL-1, i.e., that the suppression of catabolic activity in the presence of glucosamine was lost after removal of glucosamine from the medium [16]. Since the intracellular pool of glucosamine is rapidly depleted following its removal from the medium of cultured cells [21] and because furin is constitutively and continuously synthesized and recycled by cells [32], de novo furin production after removal of glucosamine would allow ADAMTS processing to occur. In addition, furin and/or other proprotein convertases secreted or shed from cells after removal of glucosamine could activate ADAMTS zymogens that were secreted unprocessed in the presence of glucosamine and deposited in the extracellular matrix [10].

The present studies contribute to understanding the previously documented protective effect of glucosamine on cartilage catabolism in vitro in conjunction with other reported effects of glucosamine such as increased transcription of aggrecan, collagen II, and the aggrecanase inhibitor TIMP3 [30, 37]. Indeed, Chan et al [38], described suppression of ADAMTS5 mRNA in bovine cartilage explants in response to glucosamine, which we did not observe in HEK293F cells. Thus glucosamine has the potential to suppress cartilage catabolism in several ways, albeit at doses that currently appear to be too high to be clinically relevant. The effect described here is of broad biological significance, since furin is responsible for molecular maturation of numerous proproteins relevant to cartilage and OA, including prohormones, growth factors, receptors, extracellular matrix proteins, and proteases [39]. Treatment with glucosamine may be useful for reversible reduction or suppression of maturation of such substrates in cell culture, since we found there were no changes in cell morphology or viability using 10 mM glucosamine.

Glucosamine is well-tolerated and is not toxic orally, but the serum and tissue concentrations achieved using the typical dose of 1500 mg/day in short-term studies are far lower than the levels we found to be required for blocking ADAMTS5 activation [35, 40]. Indeed the clinical trials testing long-term glucosamine administration do not support a slowing of arthritis progression [41, 42]. Nevertheless, the effect we report supports inhibition of aggrecanase zymogen activation as a potential therapeutic measure.


This work was supported by an award from the National Institutes of Health (AR49930) and from the Northeast Ohio Chapter of the Arthritis Foundation to S. Apte. R. Leduc is a Chercheur National of he Fonds de la Recherche en Sante du Quebec (FRSQ). We thank Dr. John Sandy for the anti-NITEGE antibody.


None of the authors have any conflict of interest relating to the submitted manuscript.

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1. Sandy JD, Flannery CR, Neame PJ, Lohmander LS. The structure of aggrecan fragments in human synovial fluid. Evidence for the involvement in osteoarthritis of a novel proteinase which cleaves the Glu 373-Ala 374 bond of the interglobular domain. J Clin Invest. 1992;89:1512–1516. [PMC free article] [PubMed]
2. Pratta MA, Yao W, Decicco C, Tortorella MD, Liu RQ, Copeland RA, et al. Aggrecan protects cartilage collagen from proteolytic cleavage. J Biol Chem. 2003;278:45539–45545. [PubMed]
3. Abbaszade I, Liu RQ, Yang F, Rosenfeld SA, Ross OH, Link JR, et al. Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family. J Biol Chem. 1999;274:23443–23450. [PubMed]
4. Tortorella MD, Burn TC, Pratta MA, Abbaszade I, Hollis JM, Liu R, et al. Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins [see comments] Science. 1999;284:1664–1666. [PubMed]
5. Fosang AJ, Little CB. Drug insight: aggrecanases as therapeutic targets for osteoarthritis. Nat Clin Pract Rheumatol. 2008;4:420–427. [PubMed]
6. Bursavich MG, Gilbert AM, Lombardi S, Georgiadis KE, Reifenberg E, Flannery CR, et al. Synthesis and evaluation of aryl thioxothiazolidinone inhibitors of ADAMTS-5 (Aggrecanase-2) Bioorg Med Chem Lett. 2007;17:1185–1188. [PubMed]
7. Glasson SS, Askew R, Sheppard B, Carito B, Blanchet T, Ma HL, et al. Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis. Nature. 2005;434:644–648. [PubMed]
8. Stanton H, Rogerson FM, East CJ, Golub SB, Lawlor KE, Meeker CT, et al. ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro. Nature. 2005;434:648–652. [PubMed]
9. Longpre JM, McCulloch DR, Koo BH, Alexander JP, Apte SS, Leduc R. Characterization of proADAMTS5 processing by proprotein convertases. Int J Biochem Cell Biol. 2009;41:1116–1126. [PubMed]
10. Malfait AM, Arner EC, Song RH, Alston JT, Markosyan S, Staten N, et al. Proprotein convertase activation of aggrecanases in cartilage in situ. Arch Biochem Biophys. 2008;478:43–51. [PubMed]
11. Wang P, Tortorella M, England K, Malfait AM, Thomas G, Arner EC, et al. Proprotein convertase furin interacts with and cleaves pro-ADAMTS4 (Aggrecanase-1) in the trans-Golgi network. J Biol Chem. 2004;279:15434–15440. [PubMed]
12. Tortorella MD, Arner EC, Hills R, Gormley J, Fok K, Pegg L, et al. ADAMTS-4 (aggrecanase-1): N-terminal activation mechanisms. Arch Biochem Biophys. 2005;444:34–44. [PubMed]
13. Sandy JD, Gamett D, Thompson V, Verscharen C. Chondrocyte-mediated catabolism of aggrecan: aggrecanase-dependent cleavage induced by interleukin-1 or retinoic acid can be inhibited by glucosamine. Biochem J. 1998;335 ( Pt 1):59–66. [PMC free article] [PubMed]
14. Dodge GR, Jimenez SA. Glucosamine sulfate modulates the levels of aggrecan and matrix metalloproteinase-3 synthesized by cultured human osteoarthritis articular chondrocytes. Osteoarthritis Cartilage. 2003;11:424–432. [PubMed]
15. Gao G, Westling J, Thompson VP, Howell TD, Gottschall PE, Sandy JD. Activation of the proteolytic activity of ADAMTS4 (aggrecanase-1) by C-terminal truncation. J Biol Chem. 2002;277:11034–11041. [PubMed]
16. Ilic MZ, Martinac B, Handley CJ. Effects of long-term exposure to glucosamine and mannosamine on aggrecan degradation in articular cartilage. Osteoarthritis Cartilage. 2003;11:613–622. [PubMed]
17. Patwari P, Gao G, Lee JH, Grodzinsky AJ, Sandy JD. Analysis of ADAMTS4 and MT4-MMP indicates that both are involved in aggrecanolysis in interleukin-1-treated bovine cartilage. Osteoarthritis Cartilage. 2005;13:269–277. [PMC free article] [PubMed]
18. Patwari P, Kurz B, Sandy JD, Grodzinsky AJ. Mannosamine inhibits aggrecanase-mediated changes in the physical properties and biochemical composition of articular cartilage. Arch Biochem Biophys. 2000;374:79–85. [PubMed]
19. Sandy JD, Thompson V, Verscharen C, Gamett D. Chondrocyte-mediated catabolism of aggrecan: evidence for a glycosylphosphatidylinositol-linked protein in the aggrecanase response to interleukin-1 or retinoic acid. Arch Biochem Biophys. 1999;367:258–264. [PubMed]
20. Elbein AD. Inhibitors of the biosynthesis and processing of N-linked oligosaccharides. CRC Crit Rev Biochem. 1984;16:21–49. [PubMed]
21. Koch HU, Schwarz RT, Scholtissek C. Glucosamine itself mediates reversible inhibition of protein glycosylation. A study of glucosamine metabolism at inhibitory concentrations in influenza-virus-infected cells. Eur J Biochem. 1979;94:515–522. [PubMed]
22. Gao G, Plaas AH, Thompson VP, Jin S, Zuo F, Sandy JD. ADAMTS4 (aggrecanase-1) activation on the cell surface involves C-terminal cleavage by GPI-anchored MT4-MMP and binding of the activated proteinase to chondroitin sulfate and heparan sulfate on syndecan-1. J Biol Chem. 2003 [PubMed]
23. Plaas A, Osborn B, Yoshihara Y, Bai Y, Bloom T, Nelson F, et al. Aggrecanolysis in human osteoarthritis: confocal localization and biochemical characterization of ADAMTS5-hyaluronan complexes in articular cartilages. Osteoarthritis Cartilage. 2007;15:719–734. [PubMed]
24. Somerville RP, Longpre JM, Jungers KA, Engle JM, Ross M, Evanko S, et al. Characterization of ADAMTS-9 and ADAMTS-20 as a distinct ADAMTS subfamily related to Caenorhabditis elegans GON-1. J Biol Chem. 2003;278:9503–9513. [PubMed]
25. Spence MJ, Sucic JF, Foley BT, Moehring TJ. Analysis of mutations in alleles of the fur gene from an endoprotease-deficient Chinese hamster ovary cell strain. Somat Cell Mol Genet. 1995;21:1–18. [PubMed]
26. Denault J, Bissonnette L, Longpre J, Charest G, Lavigne P, Leduc R. Ectodomain shedding of furin: kinetics and role of the cysteine-rich region. FEBS Lett. 2002;527:309–314. [PubMed]
27. Longpre JM, McCulloch DR, Koo BH, Alexander JP, Apte SS, Leduc R. Characterization of proADAMTS5 processing by proprotein convertases. Int J Biochem Cell Biol. 2008 [PubMed]
28. Longpre JM, Leduc R. Identification of prodomain determinants involved in ADAMTS-1 biosynthesis. J Biol Chem. 2004;279:33237–33245. [PubMed]
29. Somerville RP, Longpre JM, Apel ED, Lewis RM, Wang LW, Sanes JR, et al. ADAMTS7B, the full-length product of the ADAMTS7 gene, is a chondroitin sulfate proteoglycan containing a mucin domain. J Biol Chem. 2004;279:35159–35175. [PubMed]
30. Chan PS, Caron JP, Orth MW. Effects of glucosamine and chondroitin sulfate on bovine cartilage explants under long-term culture conditions. Am J Vet Res. 2007;68:709–715. [PubMed]
31. Derfoul A, Miyoshi AD, Freeman DE, Tuan RS. Glucosamine promotes chondrogenic phenotype in both chondrocytes and mesenchymal stem cells and inhibits MMP-13 expression and matrix degradation. Osteoarthritis Cartilage. 2007;15:646–655. [PubMed]
32. Anderson ED, Molloy SS, Jean F, Fei H, Shimamura S, Thomas G. The ordered and compartment-specfific autoproteolytic removal of the furin intramolecular chaperone is required for enzyme activation. J Biol Chem. 2002;277:12879–12890. [PMC free article] [PubMed]
33. Creemers JW, Vey M, Schafer W, Ayoubi TA, Roebroek AJ, Klenk HD, et al. Endoproteolytic cleavage of its propeptide is a prerequisite for efficient transport of furin out of the endoplasmic reticulum. J Biol Chem. 1995;270:2695–2702. [PubMed]
34. Leduc R, Molloy SS, Thorne BA, Thomas G. Activation of human furin precursor processing endoprotease occurs by an intramolecular autoproteolytic cleavage. J Biol Chem. 1992;267:14304–14308. [PubMed]
35. Laverty S, Sandy JD, Celeste C, Vachon P, Marier JF, Plaas AH. Synovial fluid levels and serum pharmacokinetics in a large animal model following treatment with oral glucosamine at clinically relevant doses. Arthritis Rheum. 2005;52:181–191. [PubMed]
36. Vlad SC, LaValley MP, McAlindon TE, Felson DT. Glucosamine for pain in osteoarthritis: why do trial results differ? Arthritis Rheum. 2007;56:2267–2277. [PubMed]
37. Toegel S, Wu SQ, Piana C, Unger FM, Wirth M, Goldring MB, et al. Comparison between chondroprotective effects of glucosamine, curcumin, and diacerein in IL-1beta-stimulated C-28/I2 chondrocytes. Osteoarthritis Cartilage. 2008;16:1205–1212. [PubMed]
38. Chan PS, Caron JP, Orth MW. Effect of glucosamine and chondroitin sulfate on regulation of gene expression of proteolytic enzymes and their inhibitors in interleukin-1- challenged bovine articular cartilage explants. Am J Vet Res. 2005;66:1870–1876. [PubMed]
39. Bergeron F, Leduc R, Day R. Subtilase-like pro-protein convertases: from molecular specificity to therapeutic applications. J Mol Endocrinol. 2000;24:1–22. [PubMed]
40. Persiani S, Rotini R, Trisolino G, Rovati LC, Locatelli M, Paganini D, et al. Synovial and plasma glucosamine concentrations in osteoarthritic patients following oral crystalline glucosamine sulphate at therapeutic dose. Osteoarthritis Cartilage. 2007;15:764–772. [PubMed]
41. Sawitzke AD, Shi H, Finco MF, Dunlop DD, Bingham CO, 3rd, Harris CL, et al. The effect of glucosamine and/or chondroitin sulfate on the progression of knee osteoarthritis: a report from the glucosamine/chondroitin arthritis intervention trial. Arthritis Rheum. 2008;58:3183–3191. [PMC free article] [PubMed]
42. Clegg DO, Reda DJ, Harris CL, Klein MA, O’Dell JR, Hooper MM, et al. Glucosamine, chondroitin sulfate, and the two in combination for painful knee osteoarthritis. N Engl J Med. 2006;354:795–808. [PubMed]
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