Binding specificity of R-10G and TRA-1-60/81, and substrate specificity of keratanase II studied with chemically synthesized oligosaccharides.

Recently, we established a mouse monoclonal antibody specific to hiPS/ hES cells, R-10G, which recognizes a type of keratan sulfate. Keratan sulfates (KS) comprise a family of glycosaminoglycans consisting of the repeating unit of [Gal-GlcNAc(6S)]. However, there is a diversity in the degree of sulfation at Gal and GlcNAc residues, and also in the mode of linkage, Galß1 - 3GlcNAc (type 1) or Galß1 - 4GlcNAc (type 2). To gain more insight into the binding specificity of R-10G, we carried out an ELISA test on avidin-coated plates using polyethylene glycol (PEG)3-biotinylated derivatives of a series of N-acetyllactosamine tetrasaccharides (keratan sulfates (KSs)). The results suggested that the minimum epitope structure is Galß1 - 4GlcNAc(6S)ß1 - 3Galß1 - 4GlcNAc(6S)ß1 (type 2- type 2 keratan sulfate). Removal of sulfate from GlcNAc(6S) or addition of sulfate to Gal abolished the binding activity almost completely. We also examined the binding specificity of TRA-1-60/81 in the same assay system. The minimum epitope structure was shown to be Galß1 - 3GlcNAcß1 - 3Galß1 - 4GlcNAcß1 in agreement with the previous study involving glycan arrays (Natunen et al., Glycobiology, 21, 1125-1130 (2011)). Interestingly, however, TRA-1-60/81 was shown to bind to Galß1 - 3GlcNAc(6S)ß1 - 3Galß1 - 4GlcNAc(6S)ß1 (type 1- type 2 keratan sulfate) dose-dependently, being more than one-third the binding activity toward Galß1 - 3GlcNAcß1 - 3Galß1 - 4GlcNAcß1 than in the case of TRA-1-60. In addition, a substrate specificity study on keratanase II revealed that keratanase II degraded not only "type 2-type 2 keratan sulfate" but also "type 1-type 2 keratan sulfate", significantly.

Microglial keratan sulfate epitope elicits in central nervous tissues of transgenic model mice and patients with amyotrophic lateral sclerosis.

The functional role of 5D4 antibody-reactive keratan sulfate (KS) in the pathogenesis of neurodegenerative diseases is unknown. We therefore studied the expression of 5D4-reactive KS in amyotrophic lateral sclerosis (ALS), a motor neuron-degenerative disease, with the use of SOD1(G93A) ALS model mice and patients with ALS. Histochemical and immunoelectron microscopic characterizations showed that the 5D4-reactive KS is expressed in Mac2/galectin-3-positive activated or proliferating microglia of SOD1(G93A) ALS model mice at disease end stage and that the KS is an O-linked glycan modified with sialic acid and fucose, which was thus far shown to exist in cartilage. Intriguingly, microglial KS was detected in the spinal cord and brainstem but not in the cerebral cortex of SOD1(G93A) mice. We found that KSGal6ST, a galactose-6-sulfotransferase, is required for biosynthesis of the microglial 5D4-reactive KS by generating SOD1(G93A)/KSGal6ST(-/-) mice. The requirement of GlcNAc6ST1 for this synthesis was corroborated by analyzing SOD1(G93A)/GlcNAc6ST1(-/-) mice. These results indicate that both galactose-6- and N acteylglucosamine-6-sulfated KS elicited in the spinal cord and brainstem are associated with the degeneration of spinal and bulbar lower motor neurons in ALS pathology and may play a role in disease progression via microglial activation and proliferation.

Generation of rat induced pluripotent stem cells using a plasmid vector and possible application of a keratan sulfate glycan recognizing antibody in discriminating teratoma formation phenotypes.

Induced pluripotent stem cells (iPSCs) offer an invaluable tool for biological research and regenerative medicine. We report establishment of rat iPSCs (riPSCs) using a plasmid vector encoding four transcription factors, Oct3/4, Sox2, c-Myc and Klf4. Although all riPSC clones were generated and cultured under the same conditions, expressed hallmark pluripotency markers and differentiated successfully in vitro, the expression of a keratan sulfate glycan epitope with unique properties defined by R-10G antibody varied in the riPSC clones. In contrast, tumor rejection antigen (TRA)-1-81 epitope expression was comparable. A clone highly reactive to R-10G antibody formed teratomas in vivo consisting of cells from all three germ layers. However, clones expressing a lower level of the epitope defined by R-10G resulted in tumors with rapid growth consisting of undifferentiated cells. Additionally, riPSCs could be successfully differentiated into a neuronal lineage including glutamate neurons that responded to agonist stimulation. These observations demonstrate a glycophenotypic difference that may potentially serve as a useful probe for riPSC evaluation and to study the role of glycans in pluripotency and carcinogenesis in these cells.

Biological interplay between proteoglycans and their innate immune receptors in inflammation.

An emerging body of evidence indicates that secreted proteoglycans act as signaling molecules, in addition to their canonical function in maintaining and regulating the architecture of various extracellular matrices. Proteoglycans interact with a number of receptors that regulate growth, motility and immune response. In part, as a consequence of their complex structure, proteoglycans can induce crosstalk among various families of receptors and can also interact with natural receptor ligands, often blocking and sequestering their bioactivity. In their soluble form, originating from either partial proteolytic processing or through de novo synthesis by activated cells, some proteoglycans can become potent danger signals, denoting tissue stress and injury. Recently, it has been shown that proteoglycans, especially those belonging to the small leucine-rich and hyaluronan-binding gene families as well as the glycosaminoglycan hyaluronan, act as endogenous ligands of the toll-like receptors, a group of central receptors regulating innate immunity. Furthermore, proteoglycans can activate intracellular inflammasomes and trigger sterile inflammation. In this review, we critically assess the signaling events induced by the proteoglycans biglycan, decorin, lumican and versican as well as hyaluronan during inflammation. We discuss the intriguing emerging notion that, in spite of structural diversity of biglycan, decorin, versican and hyaluronan, all of them signal through the same toll-like receptors, albeit triggering differential responses and biological outcomes. Finally, we review the modes of action of these endogenous ligands of toll-like receptors and their ability to specifically modify the final signaling events and the inflammatory response.

A novel antibody for human induced pluripotent stem cells and embryonic stem cells recognizes a type of keratan sulfate lacking oversulfated structures.

We have generated a monoclonal antibody (R-10G) specific to human induced pluripotent stem (hiPS)/embryonic stem (hES) cells by using hiPS cells (Tic) as an antigen, followed by differential screening of mouse hybridomas with hiPS and human embryonal carcinoma (hEC) cells. Upon western blotting with R-10G, hiPS/ES cell lysates gave a single but an unusually diffuse band at a position corresponding to >250 kDa. The antigen protein was isolated from the induced pluripotent stem (iPS) cell lysates with an affinity column of R-10G. The R-10G positive band was resistant to digestion with peptide N-glycanase F (PNGase F), neuraminidase, fucosidase, chondrotinase ABC and heparinase mix, but it disappeared almost completely on digestion with keratanase, keratanase II and endo-ß-galactosidase, indicating that the R-10G epitope is a keratan sulfate. The carrier protein of the R-10G epitope was identified as podocalyxin by liquid chromatography/mass spectrometry (LC/MS/MS) analysis of the R-10G positive-protein band material obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The R-10G epitope is a type of keratan sulfate with some unique properties. (1) The epitope is expressed only on hiPS/ES cells, i.e. not on hEC cells, unlike those recognized by the conventional hiPS/ES marker antibodies. (2) The epitope is a type of keratan sulfate lacking oversulfated structures and is not immunologically cross-reactive with high-sulfated keratan sulfate. (3) The R-10G epitope is distributed heterogeneously on hiPS cells, suggesting that a single colony of undifferentiated hiPS cells consists of different cell subtypes. Thus, R-10G is a novel antibody recognizing hiPS/ES cells, and should be a new molecular probe for disclosing the roles of glycans on these cells.

Extracellular matrix lumican promotes bacterial phagocytosis, and Lum-/- mice show increased Pseudomonas aeruginosa lung infection severity.

Phagocytosis is central to bacterial clearance, but the exact mechanism is incompletely understood. Here, we show a novel and critical role for lumican, the connective tissue extracellular matrix small leucine-rich repeat proteoglycan, in CD14-mediated bacterial phagocytosis. In Psuedomonas aeruginosa lung infections, lumican-deficient (Lum(-/-)) mice failed to clear the bacterium from lungs, tissues, and showed a dramatic increase in mortality. In vitro, phagocytosis of nonopsonized gram-negative Escherichia coli and P. aeruginosa was inhibited in Lum(-/-) peritoneal macrophages (MΦs). Lumican co-localized with CD14, CD18, and bacteria on Lum(+/+) MΦ surfaces. Using two different P. aeruginosa strains that require host CD14 (808) or CD18/CR3 (P1) for phagocytosis, we showed that lumican has a larger role in CD14-mediated phagocytosis. Recombinant lumican (rLum) restored phagocytosis in Lum(-/-) MΦs. Surface plasmon resonance showed specific binding of rLum to CD14 (K(A) = 2.15 × 10(6) M(-1)), whereas rLumY20A, and not rLumY21A, where a tyrosine in each was replaced with an alanine, showed 60-fold decreased binding. The rLumY20A variant also failed to restore phagocytosis in Lum(-/-) MΦs, indicating Tyr-20 to be functionally important. Thus, in addition to a structural role in connective tissues, lumican has a major protective role in gram-negative bacterial infections, a novel function for small leucine-rich repeat proteoglycans.

Ex vivo soft-laser treatment inhibits the synovial expression of vimentin and α-enolase, potential autoantigens in rheumatoid arthritis.

BACKGROUND: Soft-laser therapy has been used to treat rheumatic diseases for decades. The major effects of laser treatment may be dependent not on thermal mechanisms but rather on cellular, photochemical mechanisms. However, the exact cellular and molecular mechanisms of action have not been elucidated. OBJECTIVE: The aim of this study was to investigate the ex vivo effects of low-level laser treatment (with physical parameters similar to those applied previously) on protein expression in the synovial membrane in rheumatoid arthritis (RA). DESIGN: Synovial tissues were laser irradiated, and protein expression was analyzed. METHODS: Synovial membrane samples obtained from 5 people who had RA and were undergoing knee surgery were irradiated with a near-infrared diode laser at a dose of 25 J/cm(2) (a dose used in clinical practice). Untreated synovial membrane samples obtained from the same people served as controls. Synovial protein expression was assessed with 2-dimensional polyacrylamide gel electrophoresis followed by mass spectrometry. RESULTS: The expression of 12 proteins after laser irradiation was different from that in untreated controls. Laser treatment resulted in the decreased expression of α-enolase in 2 samples and of vimentin and precursors of haptoglobin and complement component 3 in 4 samples. The expression of other proteins, including 70-kDa heat shock protein, 96-kDa heat shock protein, lumican, osteoglycin, and ferritin, increased after laser therapy. LIMITATIONS: The relatively small sample size was a limitation of the study. CONCLUSIONS: Laser irradiation (with physical parameters similar to those used previously) resulted in decreases in both α-enolase and vimentin expression in the synovial membrane in RA. Both proteins have been considered to be important autoantigens that are readily citrullinated and drive autoimmunity in RA. Other proteins that are expressed differently also may be implicated in the pathogenesis of RA. Our results raise the possibility that low-level laser treatment of joints affected with RA may be effective, at least in part, by suppressing the expression of autoantigens. Further studies are needed.

Lumican is required for neutrophil extravasation following corneal injury and wound healing.

An important aspect of wound healing is the recruitment of neutrophils to the site of infection or tissue injury. Lumican, an extracellular matrix component belonging to the small leucine rich proteoglycan (SLRP) family, is one of the major keratan sulfate proteoglycans (KSPGs) within the corneal stroma. Increasing evidence indicates that lumican can serve as a regulatory molecule for several cellular processes, including cell proliferation and migration. In the present study, we addressed the role of lumican in the process of extravasation of polymorphonuclear leukocytes (PMNs) during the early inflammatory phase present in the healing of the corneal epithelium following debridement. We used Lum(-/-) mice and a novel transgenic mouse, Lum(-/-),Kera-Lum, which expresses lumican only in the corneal stroma, to assess the role of lumican in PMN extravasation into injured corneas. Our results showed that PMNs did not readily invade injured corneas of Lum(-/-) mice and this defect was rescued by the expression of lumican in the corneas of Lum(-/-),Kera-Lum mice. The presence of lumican in situ facilitates PMN infiltration into the peritoneal cavity in casein-induced inflammation. Our findings are consistent with the notion that in addition to regulating the collagen fibril architecture, lumican acts to aid neutrophil recruitment and invasion following corneal damage and inflammation.

Extracellular matrix lumican deposited on the surface of neutrophils promotes migration by binding to beta2 integrin.

During inflammation, circulating polymorphonuclear neutrophils (PMNs) receive signals to cross the endothelial barrier and migrate through the extracellular matrix (ECM) to reach the injured site. Migration requires complex and poorly understood interactions of chemokines, chemokine receptors, ECM molecules, integrins, and other receptors. Here we show that the ECM protein lumican regulates PMN migration through interactions with specific integrin receptors. Lumican-deficient (Lum(-/-)) mice manifest connective tissue defects, impaired innate immune response, and poor wound healing with reduced PMN infiltration. Lum(-/-) PMNs exhibit poor chemotactic migration that is restored with exogenous recombinant lumican and inhibited by anti-lumican antibody, confirming a role for lumican in PMN migration. Treatment of PMNs with antibodies that block beta(2), beta(1), and alpha(M) integrin subunits inhibits lumican-mediated migration. Furthermore, immunohistochemical and biochemical approaches indicate binding of lumican to beta(2), alpha(M), and alpha(L) integrin subunits. Thus, lumican may regulate PMN migration mediated by MAC-1 (alpha(M)/beta(2)) and LFA-1 (alpha(L)/beta(2)), the two major PMN surface integrins. We detected lumican on the surface of peritoneal PMNs and not bone marrow or peripheral blood PMNs. This suggests that PMNs must acquire lumican during or after crossing the endothelial barrier as they exit circulation. We also found that peritoneal PMNs do not express lumican, whereas endothelial cells do. Taken together these observations suggest a novel endothelial lumican-mediated paracrine regulation of neutrophils early on in their migration path.

Immunophenotypes of macular corneal dystrophy in India and correlation with mutations in CHST6.

PURPOSE: To determine the immunophenotypes of macular corneal dystrophy (MCD) in Indian patients and to correlate them with mutations in the carbohydrate 6-sulfotransferase (CHST6) gene. METHODS: Sixty-four patients from 53 families with MCD that were previously screened for mutations in CHST6 were included in an immunophenotype analysis. Antigenic keratan sulfate (AgKS) in serum as well as corneal tissue was evaluated in 31 families. Only cornea was evaluated in 11 families, and only serum was evaluated in 11 families. AgKS was detected in formalin-fixed, paraffin-embedded corneal sections by immunohistochemistry and in serum by ELISA using a monoclonal antibody against sulfated forms of KS in patients with MCD as well as normal controls. RESULTS: Analysis of corneal and/or serum AgKS disclosed MCD type I (27 families), MCD type IA (5 families), and MCD type II (3 families) in the cases studied. An additional 10 families were either MCD type I or MCD type IA since only serum AgKS data were available. Seven families manifested atypical immunophenotypes since the corneal AgKS expression was either of MCD type I or MCD type IA, but serum AgKS levels ranged from 19 ng/ml to 388 ng/ml. More than one immunophenotype was detected amongst siblings in two families. Each immunophenotype was associated with mutational heterogeneity in CHST6. CONCLUSIONS: MCD type I was the predominant immunophenotype in the Indian population studied followed by MCD type IA and then MCD type II. We detected further immunophenotypic heterogeneity by finding atypical patterns of AgKS reactivity in a subset of families. There were no simple correlations between immunophenotypes and specific mutations in CHST6, suggesting that factors other than CHST6 mutations may be contributing to the immunophenotypes in MCD.

Application of proteomics in the discovery of candidate protein biomarkers in a diabetes autoantibody standardization program sample subset.

Novel biomarkers of type 1 diabetes must be identified and validated in initial, exploratory studies before they can be assessed in proficiency evaluations. Currently, untargeted "-omics" approaches are underutilized in profiling studies of clinical samples. This report describes the evaluation of capillary liquid chromatography (LC) coupled with mass spectrometry (MS) in a pilot proteomic analysis of human plasma and serum from a subset of control and type 1 diabetic individuals enrolled in the Diabetes Autoantibody Standardization Program, with the goal of identifying candidate biomarkers of type 1 diabetes. Initial high-resolution capillary LC-MS/MS experiments were performed to augment an existing plasma peptide database, while subsequent LC-FTICR studies identified quantitative differences in the abundance of plasma proteins. Analysis of LC-FTICR proteomic data identified five candidate protein biomarkers of type 1 diabetes. alpha-2-Glycoprotein 1 (zinc), corticosteroid-binding globulin, and lumican were 2-fold up-regulated in type 1 diabetic samples relative to control samples, whereas clusterin and serotransferrin were 2-fold up-regulated in control samples relative to type 1 diabetic samples. Observed perturbations in the levels of all five proteins are consistent with the metabolic aberrations found in type 1 diabetes. While the discovery of these candidate protein biomarkers of type 1 diabetes is encouraging, follow up studies are required for validation in a larger population of individuals and for determination of laboratory-defined sensitivity and specificity values using blinded samples.

Sequencing of the CHST6 gene in Czech macular corneal dystrophy patients supports the evidence of a founder mutation.

AIMS: To characterise the role of the carbohydrate sulfotransferase gene (CHST6) in macular corneal dystrophy (MCD) in Czech patients. METHODS: The coding region of the CHST6 gene was directly sequenced in 10 affected and five unaffected members from eight apparently unrelated MCD families. The type of MCD was determined by enzyme-linked immunosorbent assay of antigenic keratan sulfate (KS) in serum and by immunohistochemical staining of corneas with monoclonal anti-KS antibody. RESULTS: The following changes in the coding sequence of the CHST6 gene were observed; homozygous mutation of c.1A>T (p.M1?); homozygous mutation c.599T>G (p.L200R); compound heterozygosity for c.599T>G and c.614G>A (p.R205Q); compound heterozygosity for c.494G>A (p.C165Y) and c.599T>G; heterozygous c.599T>G mutation and no other change in the coding sequence. One proband exhibited no changes. The pathogenic mutation c.599T>G (p.L200R) was in allelic association with the c.484C>G (p.R162G) polymorphism. Nine patients from seven families were of MCD type I including the subtype IA. CONCLUSION: Four different CHST6 missense mutations, of which p.C165Y is novel, were identified. Allelic association of the c.[484C>G; 599T>G] in six probands out of eight, as well as occurrence of this particular allele in a heterozygous state in one healthy control individual, supports a common founder effect for MCD in the Czech Republic.

A novel role of the lumican core protein in bacterial lipopolysaccharide-induced innate immune response.

Lumican is an extracellular matrix protein modified as a proteoglycan in some tissues. The core protein with leucine-rich repeats, characteristic of the leucine-rich-repeat superfamily, binds collagen fibrils and regulates its structure. In addition, we believe that lumican sequestered in the pericellular matrix interacts with cell surface proteins for specific cellular functions. Here we show that bacterial lipopolysaccharide sensing by the Toll-like receptor 4 signaling pathway and innate immune response is regulated by lumican. Primary cultures of lumican-deficient (Lum(-/-)) macrophages show impaired innate immune response to lipopolysaccharides with lower induction of tumor necrosis factor alpha (TNFalpha) and interleukin-6. Macrophage response to other pathogen-associated molecular patterns is not adversely affected by lumican deficiency, suggesting a specific role for the lumican core protein in the Toll-like receptor 4 pathway. An exogenous recombinant lumican core protein increases lipopolysaccharide-mediated TNFalpha induction and partially rescues innate immune response in Lum(-/-) macrophages. We further show that the core protein binds lipopolysaccharide. Immunoprecipitation of lumican from peritoneal lavage co-precipitates CD14, a cell surface lipopolysaccharide-binding protein that is involved in its presentation to Toll-like receptor 4. The Lum(-/-) mice are hypo-responsive to lipopolysaccharide-induced septic shock, with poor induction of pro-inflammatory cytokines, TNFalpha, and interleukins 1beta and 6 in the serum. Taken together, the data indicates a novel role for lumican in the presentation of bacterial lipopolysaccharide to CD14 and host response to this bacterial endotoxin.

Nicotine downregulates the l-selectin system that mediates cytotrophoblast emigration from cell columns and attachment to the uterine wall.

Here we show that maternal smoking downregulated, in a dose-dependent manner, cytotrophoblast expression of l-selectin and its TRA-1-81-reactive carbohydrate ligands. Cell islands -- cell columns that fail to make uterine attachments, often more numerous in the placentas of smokers -- exhibited an even greater downregulation of the l-selectin adhesion system. These effects were attributable to nicotine, since exposure of explanted villi to this drug in vitro reproduced the effects observed in situ. Videomicroscopy showed that the downstream consequences included inhibition of all stages of cytotrophoblast outgrowth from columns, including rolling adhesion within columns and generation of invasive cells at the distal ends. These results suggest that nicotine, acting through the l-selectin adhesion system, impairs the development of cell columns that connect the fetal portion of the placenta to the uterus, one possible reason why women who smoke have a much harder time achieving and sustaining pregnancy than their nonsmoking counterparts.

Osteogenic protein 1 in synovial fluid from patients with rheumatoid arthritis or osteoarthritis: relationship with disease and levels of hyaluronan and antigenic keratan sulfate.

The measurement of body fluid levels of biochemical markers in joint tissues has begun to provide clinically useful information. Synovial fluid (SF) plays an important role in articular joint lubrication, nutrition, and metabolism of cartilage and other connective tissues within the joint. The purpose of our study was to identify and characterize osteogenic protein 1 (OP-1) in SF from patients with rheumatoid arthritis (RA) or with osteoarthritis (OA) and to correlate levels of OP-1 with those of hyaluronan (HA) and antigenic keratan sulfate (AgKS). SF was aspirated from the knees of patients with either RA or OA and from the knees of asymptomatic organ donors with no documented history of joint disease. The presence of detectable OP-1 in SF was demonstrated by western blots with specific anti-pro-OP-1 and anti-mature OP-1 antibodies. Measurement of levels of OP-1, HA and AgKS was performed using ELISAs. OP-1 was identified in human SF in two forms, pro-OP-1 and active (mature) OP-1--mature OP-1 being detected only in SF from OA patients and RA patients. Levels of OP-1 and HA were higher in RA patients than in OA patients and asymptomatic donors, while the level of AgKS was highest in SF from asymptomatic donors. Statistically significant differences were found between SF levels of OP-1 in RA and OA patients and between SF levels of AgKS among the three groups tested. The SF content of OP-1 tended to correlate positively with HA levels, but negatively with AgKS concentrations. In conclusion, the results of this study suggest that measurement of OP-1 in joint fluid may have value in the clinical evaluation of joint disease processes.

Use of biochemical markers of osteoarthritis to investigate the potential disease-modifying effect of tibial plateau levelling osteotomy.

OBJECTIVES: To evaluate the hypothesis that the concentration of the 1/20/5D4 epitope of keratan sulphate, cartilage oligomeric matrix protein and total sulphated glycosaminoglycans in synovial fluids from dogs with cranial cruciate ligament disease would be affected by tibial plateau levelling osteotomy. In addition, to evaluate the hypothesis that medial meniscal release or meniscal injury would alter the expression of these candidate biomarkers. METHODS: Forty-one dogs with naturally occurring cranial cruciate ligament disease were recruited prospectively. Synovial fluids were collected from the index joint before surgery and six weeks and six months postsurgery. Following tibial plateau levelling osteotomy, synovial fluids were assayed for 1/20/5D4 epitope of keratan sulphate and cartilage oligomeric matrix protein concentration using an inhibition ELISA and for sulphated glycosaminoglycans using a direct dye-binding assay. RESULTS: The sulphated glycosaminoglycans ratio did not change significantly during the study. Medial meniscal injury at entry was associated with lower concentrations of synovial fluid cartilage oligomeric matrix protein (P<0.05, unpaired t test). There was no association between medial meniscal release and the changes in marker concentrations, either from 0 to six weeks or 0 to six months. CLINICAL SIGNIFICANCE: Tibial plateau levelling osteotomy did not significantly alter the expression of the named candidate biomarkers. These findings reflect the limited nature of the arthrotomy or indicate that tibial plateau levelling osteotomy does not influence the progression of osteoarthritis (OA). From these studies, there is no evidence that tibial plateau levelling osteotomy affects cartilage metabolism.

Keratan sulphate levels in mucopolysaccharidoses and mucolipidoses.

The mucopolysaccharidoses (MPS) is characterized by accumulation of glycosaminoglycans (GAGs), and mucolipidosis (ML) by accumulation of GAGs and sphingolipids. Each type of MPS accumulates specific GAGs. The lysosomal enzymes N-acetylgalactosamine-6-sulphate sulphatase and beta-galactosidase involve the stepwise degradation of keratan sulphate (KS). Deficiency of these enzymes results in elevation of KS levels in the body fluids and in tissues, leading to MPS IV disease. In this study, we evaluated blood and urine KS levels in types of MPS and ML other than MPS IV. Eighty-five plasma samples came from MPS I (n = 18), MPS II (n = 28), MPS III (n = 20), MPS VI (n = 3), MPS VII (n = 5) and ML (n = 11) patients while 127 urine samples came from MPS I (n = 34), MPS II (n = 34), MPS III (n = 32), MPS VI (n = 7), MPS VII (n = 9) and ML (n = 11) patients. KS levels were determined using the ELISA method. Plasma KS levels varied with age in both control and patient populations. In all age groups, the mean values of plasma KS in MPS and ML patients were significantly higher than those in the age-matched controls. Plasma KS values in four newborn patients were above the mean + 2SD of the age-matched controls (mean, 41 ng/ml). Overall, 85.9% of individual values in non-type IV MPS and ML patients were above the mean + 2SD of the age-matched controls. For urine KS levels, 24.4% of individual values in patients were above the mean + 2SD of the age-matched controls. In conclusion, KS in blood is elevated in each type of non-type IV MPS examined, in contrast to the conventional understanding. This finding suggests that measurement of KS level provides a new diagnostic biomarker in a wide variety of mucopolysaccharidoses and mucolipidoses in addition to MPS IV.

Monoclonal antibody 4C4-mAb specifically recognizes keratan sulphate proteoglycan on human embryonal carcinoma cells.

Germ cell tumours, the most common solid cancers in young males, display pluripotentiality for embryonal and somatic differentiation. Specific surface antigens are useful in the study of cellular differentiation and for clinical diagnosis. A mouse monoclonal antibody (4C4-mAb) has been developed against a human embryonal carcinoma (EC) cell line (NCR-G3) isolated from a combined form of testicular germ cell tumour. On immunohistological and immuno-electron microscopic examination, the 4C4 antigen (4C4) was detected on the surface of NCR-G3 and gold particles were exclusively detected on the microvilli of the cells. In both formalin-fixed paraffin wax sections and touch-smear specimens, 4C4 was detected specifically in EC, while the antigen was not expressed in other types of germ cell tumour or in the other solid tumours tested. Tunicamycin diminished the antigenicity of NCR-G3 cells. In biochemical studies, 4C4 was found in a high molecular weight region ranging from 1 x 10(6) to 1 x 10(7) kD, which disappeared after periodate treatment. The density of 4C4 was 1.5 g/cm(3) after equilibrium centrifugation. These results imply that 4C4 is a proteoglycan. Furthermore, endo- and exo-glycosidase treatment revealed that 4C4 is a keratan sulphate proteoglycan that contains sialyl and fucosyl moieties. With EC-specific and formalin-resistant characteristics, 4C4 may be a specific marker for diagnosing EC among a variety of germ cell tumours.

Detection of collagen type II and proteoglycans in the synovial fluids of patients diagnosed with non-infectious knee joint synovitis indicates early damage to the articular cartilage matrix.

OBJECTIVE: We have sought to determine if markers of proteoglycans and collagen type II (CII) degradation can be detected at an early stage following acute knee injury in the synovial fluid (SF) from a group of patients diagnosed with non-infectious knee joint synovitis (KJS). CII, proteoglycans and elastase activity in the SF from patients with KJS were compared to SF from patients with two chronic arthritis conditions: osteoarthritis (OA) and rheumatoid arthritis (RA) as well as normal SF controls. METHODS: CII peptides were measured by sandwich ELISA using two monoclonal antibodies: 8:6:D8, a CII-specific antibody, and 14:7:D8 which binds to an amino acid sequence on CII as well as collagens type I, III and V. Epitope 9A4, a neo-epitope resulting from collagenase digestion of CI, CII, and CIII was measured by inhibition ELISA. Proteoglycans measurement included total sulfated glycosaminoglycans (sGAG) by dye-binding assay and 5-D-4 epitope, a keratan sulfate epitope, by inhibition ELISA. Elastase activity was measured colorimetircally using N-succinyl trialanine p-nitroanilide (SANA) substrate. RESULTS: The quantified CII peptide concentrations by sandwich and inhibition ELISA were significantly higher in SF from patients with KJS (P<0.05) compared to SF from patients with OA, RA and normal aspirates. 5-D-4 and sGAG concentrations were significantly lower (P<0.05) in SF from patients with KJS compared to SF from patients with OA and RA. Elastase activity in SF from patients with KJS and RA were significantly higher (P<0.05) than SF from patients with OA. A significant correlation exists between elastase activity and 9A4 epitope concentration in SF from patients with KJS. CONCLUSION: The elevated CII peptides concentrations in KJS SF compared to normal and OA aspirates indicate early signs of cartilage network damage. The low proteoglycans concentrations in SF from patients with KJS may indicate that injury is limited to the superficial zone of cartilage in the patient population studied. The high elastase activity in SF from patients with KJS and RA are linked to the high CII peptides concentration. The elastase activity in the SF from patients with KJS is due to the action of neutrophil elastase (NE) and collagenases, where both contribute to the destruction of the articular cartilage.

Increased expression of the putative axon growth-repulsive extracellular matrix molecule, keratan sulphate proteoglycan, following traumatic injury of the adult rat spinal cord.

Keratan sulphate proteoglycan (KSPG) is a developmentally regulated barrier molecule, directing axonal growth during central nervous system (CNS) formation. The possible re-expression and functional significance of KSPG in preventing axon regeneration following spinal cord injury (SCI) is poorly understood. In the present investigation, the spatio-temporal expression of KSPG was studied following experimental SCI. There was no indication of sparing of axons at the lesion epicentre following severe compression injury. By 7 days post operation (p.o.) a diffuse increase of KSPG immunoreactivity (KSPG-IR) was observed in the parenchyma surrounding the lesion. This was followed by a delayed (21-28 days p.o.) and largely heterogeneous increase of KSPG-IR in the lesion epicentre, which revealed both cellular and extracellular matrix-like distribution patterns. Although no re-growth of anterogradely labelled corticospinal axons was observed, many 200-kDa neurofilament (NF)-positive axons could be detected growing into the connective tissue scar. This phase of spontaneous axonal re-growth was closely associated with a framework of glial cells (including Schwann cells from damaged local spinal nerve roots) that had migrated into the lesion site. The spontaneous nerve fibre re-growth could be detected in both KSPG-rich and KSPG-poor territories. The present data suggest that the lesion-induced up-regulation of KSPG-IR may have contributed to the lack of corticospinal axon re-growth. However, the lack of any direct spatio-temporal correlation between the distribution of raised KSPG-IR and spontaneous NF-positive axonal regeneration suggests that at least some populations of axons can resist the putative inhibitory effects of this extracellular matrix molecule.