Synovial fluid level of aggrecan ARGS fragments is a more sensitive marker of joint disease than glycosaminoglycan or aggrecan levels: a cross-sectional study

Introduction Aggrecanase cleavage at the (392)Glu-(393)Ala bond in the interglobular domain (IGD) of aggrecan, releasing N-terminal (393)ARGS fragments, is an early key event in arthritis and joint injuries. Here, we use a quantitative immunoassay of aggrecan ARGS neoepitope fragments in human synovial fluid to determine if this cleavage-site specific method better identifies joint pathology than previously available less specific aggrecan assays. Methods Synovial fluid (SF) from 26 people with healthy knees (reference) and 269 patients were analyzed in a cross-sectional study. Patient groups were acute inflammatory arthritis, acute knee injury, chronic knee injury and knee osteoarthritis (OA). Aggrecan ARGS fragments were assayed by ELISA using the monoclonal antibody OA-1. Total aggrecan content was analyzed by an ELISA using the monoclonal antibody 1-F21, and sulfated glycosaminoglycan by Alcian blue precipitation. Results Aggrecan ARGS fragment concentrations in all groups differed from the reference group (P < 0.001). The acute inflammatory arthritis group had the highest median level, 177-fold greater than that of the reference group. Median levels (in pmol ARGS/ml SF) were: reference 0.5, acute inflammatory arthritis 88.5, acute knee injury 53.9, chronic knee injury 0.5 and OA 4.6. In contrast, aggrecan and sulfated glycosaminoglycan concentrations varied much less between groups, and only acute inflammatory arthritis and acute knee injury were found to have a two-fold increase in median levels compared to the reference. Conclusions Levels of aggrecan ARGS fragments in human synovial fluid are increased in human arthritis, OA and after knee injury, likely reflecting an enhanced cleavage at the (392)Glu-(393)Ala bond in the IGD by aggrecanase. An assay that specifically quantified these fragments better distinguished samples from joints with pathology than assays monitoring aggrecan or glycosaminoglycan concentrations. The newly developed ARGS fragment assay can be used to monitor aggrecanase activity in human joint disease and experimental models

Association between synovial fluid levels of aggrecan ARGS fragments and radiographic progression in knee osteoarthritis

INTRODUCTION: Aggrecanase cleavage at the (392)Glu-(393)Ala bond in the interglobular domain (IGD) of aggrecan, releasing N-terminal (393)ARGS fragments, is an early key event in arthritis and joint injuries. We determined whether synovial fluid (SF) levels of ARGS-aggrecan distinguish subjects with progressive radiographic knee osteoarthritis (ROA) from those with stable or no ROA. METHODS: We studied 141 subjects who, at examination A, had been given meniscectomies an average of 18 years earlier (range, 15 to 22 years). Seventeen individuals without surgery, and without known injury to the menisci or cruciate ligaments, were used as references. At examinations A and B, with a mean follow-up time of 7.5 years, we obtained SF and standing tibiofemoral and skyline patellofemoral radiographs. SF ARGS-aggrecan was measured with an electrochemiluminescence immunoassay, and we graded radiographs according to the OARSI atlas. The association between SF ARGS levels at examination A and progression of radiographic features of knee OA between examinations A and B was assessed by using logistic regression adjusted for age, gender, body mass index, and time between examinations, and stratified by ROA status at examination A. RESULTS: We found a weak negative association between SF ARGS concentrations and loss of joint space: the likelihood of progression of radiographic joint space narrowing decreased 0.9 times per picomole per milliliter increase in ARGS (odds ratio (OR) 0.89; 95% confidence interval (CI), 0.79 to 0.996). In subjects with and without preexisting ROA at examination A, the association was OR, 0.96; 0.81 to 1.13; and 0.77; 0.62 to 0.95, respectively. Average levels of SF ARGS 18 years after meniscectomy were no different from those of reference subjects and were not correlated to radiographic status at examination A. CONCLUSIONS: In subjects with previous knee meniscectomy but without ROA, levels of SF ARGS-aggrecan were weakly and inversely associated with increased loss of joint space over a period of 7.5 years

Protein phosphorylation/dephosphorylation in the inner membrane of potato tuber mitochondria

AbstractInside-out inner mitochondrial membranes free of matrix proteins were isolated from purified potato tuber (Solanum tuberosum L.) mitochondria and incubated with [γ-32P]ATP. Proteins were separated by SDS–PAGE and visualized by autoradiography. Phosphorylation of inner membrane proteins, including ATPase subunits, was strongly inhibited by the phosphoprotein phosphatase inhibitor NaF. We propose that an inner membrane phosphoprotein phosphatase is required for activation of the inner membrane protein kinase. When prelabelled inner membranes were incubated in the absence of [γ-32P]ATP, there was no phosphoprotein dephosphorylation unless a soluble matrix fraction was added. This dephosphorylation was inhibited by NaF, but not by okadaic acid. We conclude that the mitochondrial matrix contains a phosphoprotein phosphatase that is responsible for dephosphorylation of inner membrane phosphoproteins

The complement system is activated in synovial fluid from subjects with knee injury and from patients with osteoarthritis

Concentrations of C4d, C3bBbP and sTCC in synovial fluid. (DOCX 37 kb

Coculture of bovine cartilage with synovium and fibrous joint capsule increases aggrecanase and matrix metalloproteinase activity

Background A hallmark of osteoarthritis is increased proteolytic cleavage of aggrecan. Cross talk between cartilage and the synovium + joint capsule (SJC) can drive cartilage degradation by activating proteases in both tissues. We investigated aggrecan proteolysis patterns in cartilage explants using a physiologically relevant explant model of joint injury combining cartilage mechanical compression and coincubation with SJC. Methods Bovine cartilage explants were untreated; coincubated with SJC; or subjected to mechanical injury and coincubated with SJC, mechanical injury alone, or mechanical injury and incubated with tumor necrosis factor-α (TNF-α). To compare the patterns of aggrecan proteolysis between 6 h and 16 days, release of sulfated glycosaminoglycans and specific proteolytic aggrecan fragments into medium or remaining in cartilage explants was measured by dimethylmethylene blue and Western blot analysis. Results Aggrecanase activity toward aggrecan was observed in all conditions, but it was directed toward the TEGE↓ARGS interglobular domain (IGD) site only when cartilage was coincubated with SJC or TNF-α. Matrix metalloproteinase (MMP) activity at the aggrecan IGD site (IPES↓FFGV) was not detected when cartilage was exposed to TNF-α (up to 6 days), but it was in all other conditions. Compared with when bovine cartilage was left untreated or subjected to mechanical injury alone, additional aggrecan fragment types were released into medium and proteolysis of aggrecan started at an earlier time when SJC was present. Conclusions Indicative of different proteolytic pathways for aggrecan degradation, the SJC increases both aggrecanase and MMP activity toward aggrecan, whereas TNF-α inhibits MMP activity against the IGD of aggrecan.National Institutes of Health (U.S.) (AR060331

Protein phosphorylation in plant mitochondria

Protein phosphorylation in the subcompartments of plant mitochondria was investigated by labelling with [gamma-32P]ATP and by SDS-PAGE/autoradiography. About 20 proteins in inside-out inner mitochondrial membranes from potato tubers were phosphorylated by endogenous protein kinases when incubated with [gamma-32P]ATP. None of the phosphorylated proteins were labelled when using [alpha-32P]ATP as a phosphate donor. The reversible protein phosphorylation in these membranes is mainly a result of the action of inner membrane serine/threonine protein kinases/phosphatases, although the dephosphorylation of inner membrane proteins appears to be catalysed by protein phosphatases located in the matrix. In addition, a 37 kDa protein contained acid-labile phosphate groups, indicating modification of histidine residues. The phosphorylation of inner membrane proteins requires divalent cations (i.e. Mg2+ or Mn2+) and is stimulated under oxidising conditions. Two autophosphorylated inner membrane proteins with molecular mass of 16.5 and 30 kDa are putative protein kinases. Two inner membrane phosphoproteins with molecular mass of 22 and 28 kDa were identified by N-terminal sequencing as the delta?-subunit and b-subunit, respectively, of the FoF1-ATPase. A 17 kDa phosphoprotein has some characteristics of a nucleoside diphosphate kinase. All other plant inner mitochondrial membrane phosphoproteins remain to be identified. A 39.5 and 41 kDa protein are the most heavily labelled phosphoproteins in the matrix of potato tuber mitochondria. The 41 kDa protein is probably the alpha-subunit of pyruvate dehydrogenase, and the 39.5 kDa protein, which was also labelled with 32Pi, may be the alpha-subunit of succinyl-CoA synthetase. In pea leaf mitochondria, a 17.4 kDa protein, which is probably located in the intermembrane space, was purified and identified as a nucleoside diphosphate kinase. This pea protein is autophosphorylated on both the catalytic histidine and on serine residues. The labelling on serine residues was higher then for the histidine, and kinetic studies demonstrated a faster rate of phosphorylation of serine compared to histidine

Calpain is involved in C-terminal truncation of human aggrecan

Mature aggrecan is generally C-terminally truncated at several sites in the CS (chondroitin sulfate) region. Aggrecanases and MMPs (matrix metalloproteinases) have been suggested to be responsible for this digestion. To identify whether calpain, a common intracellular protease, has a specific role in the proteolysis of aggrecan we developed neoepitope antibodies (anti-PGVA, anti-GDLS and anti-EDLS) against calpain cleavage sites and used Western blot analysis to identify alpain-generated fragments in normal and OA (osteoarthritis) knee cartilage and SF (synovial fluid) samples. Our results showed that human aggrecan contains six calpain cleavage sites: one in the IGD (interglobular domain), one in the KS (keratan sulfate) region, two in the CS1 and two in the CS2 region. Kinetic studies of calpain proteolysis against aggrecan showed that the aggrecan molecule was cleaved in a specific order where cuts in CS1 was the most preferred and cuts in KS region was the second most preferred cleavage. OA and normal cartilage contained low amounts of a calpain-generated G1-PGVA fragment (0.5-2%) compared with aggrecanase-generated G1-TEGE (71-76%) and MMP-generated G1-IPEN (23-29%) fragments. Significant amounts of calpain-generated GDLS and EDLS fragments were found in OA and normal cartilage, and a ARGS-EDLS fragment was detected in arthritic SF samples. The results of the present study indicate that calpains are involved in the C-terminal truncation of aggrecan and might have a minor role in arthritic diseases

A comparison of different purification methods of aggrecan fragments from human articular cartilage and synovial fluid.

In the study of aggrecan fragmentation several methods to extract and purify aggrecan from cartilage and synovial fluid (SF) are used. This work compares and evaluates the effectiveness for purification of aggrecan of the most commonly used methods by the ratio of sulfated glycosaminoglycan (sGAG) to protein and by fragment analysis by Western blot. A novel method for purification of aggrecan fragments from SF by boiling (Boiled SF) is also presented. Of the sGAG extracted from cartilage by guanidinium, 66% was recovered by associative-dissociative cesium chloride density gradient centrifugation (A1D1-D3) with a 9 times higher ratio of sGAG to protein in the A1D1 fraction. Although less enriched in aggrecan, the Western blot aggrecan pattern of the guanidinium extracted sample resembled that of the combined patterns of the A1D1, A1D2 and A1D3 fractions. The recoveries of sGAG from SF purified by anion chromatography and Alcian blue precipitation were around 50%, while the recoveries were over 80% in the associative or dissociative density gradient fractions (A1 and D1) and Boiled SF. The purification compared to neat SF ranged from 9 times in boiled SF to 1800-1900 times in Alcian blue and D1 samples. To obtain reliable results when analyzing synovial fluid aggrecan fragments by Western blot, purification was necessary. The immuno-pattern of anion chromatography purified SF resembled the patterns of A1 and D1, while the pattern of Boiled SF resembled the D1 sample. This work suggests that aggrecan fragments extracted from cartilage by guanidinium need no further purification to be analyzed by Western blot, whereas aggrecan fragments in SF are best analyzed in the A1 and D1 fractions or in the Boiled SF sample

Purification of a serine and histidine phosphorylated mitochondrial nucleoside diphosphate kinase from Pisum sativum

For the first time, to our knowledge, a nucleoside diphosphate kinase (NDPK) has been purified from plant mitochondria (Pisum sativum L.). In intact pea leaf mitochondria, a 17.4-kDa soluble protein was phosphorylated in the presence of EDTA when [γ-32]ATP was used as the phosphate donor. Cell fractionation demonstrated that the 17.4-kDa protein is a true mitochondrial protein, and the lack of accessibility to EDTA of the matrix compartment in intact mitochondria suggested it may have an intermembrane space localization. The 17.4-kDa protein was purified from mitochondrial soluble proteins using ATP-agarose and anion exchange chromatography. Amino- acid sequencing of two peptides, resulting from a trypsin digestion, revealed high similarity with the conserved catalytic phosphohistidine site and with the C-terminal of NDPKs. Acid and alkali treatments of [32P]-labelled pea mitochondrial NDPK indicated the presence of acid-stable as well as alkali- stable phosphogroups. Thin-layer chromatography experiments revealed serine as the acid-stable phosphogroup. The alkali-stable labelling probably reflects phosphorylation of the conserved catalytic histidine residue. In phosphorylation experiments, the purified pea mitochondrial NDPK was labelled more heavily on serine than histidine residues. Furthermore, kinetic studies showed a faster phosphorylation rate for serine compared to histidine. Both ATP and GTP could be used as phosphate donor for histidine as well as serine labelling of the pea mitochondrial NDPK