Effect of circadian rhythm, age, training and acute lameness on serum concentrations of cartilage oligomeric matrix protein (COMP) neo-epitope in horses.

BACKGROUND: Molecular serum markers that can identify early reversible osteoarthritis (OA) in horses are lacking. OBJECTIVES: We studied serum concentrations of a novel cartilage oligomeric matrix protein (COMP) neo-epitope in horses subjected to short-term exercise and with acute lameness. The effects of circadian rhythm and age were also evaluated. STUDY DESIGN: Longitudinal studies in healthy horses and cross-sectional comparison of lame and non-lame horses. METHODS: Sera were collected from five horses before and after short-term interval exercise and during full-day box rest. Sera from 32 acutely lame horses were used to evaluate age-related effects. Independent samples from control horses (n = 41) and horses with acute lameness (n = 71) were included. COMP neo-epitope concentrations were analysed using custom-developed inhibition ELISAs validated for equine serum. The presence of COMP neo-epitope was delineated in healthy and osteoarthritic articular cartilage with immunohistochemistry. RESULTS: COMP neo-epitope concentrations decreased after speed training but returned to baseline levels post-exercise. No correlations between age and serum COMP neo-epitope concentrations were found (r = 0.0013). The mean (±s.d.) serum concentration of COMP neo-epitope in independent samples from non-lame horses was 0.84 ± 0.38 µg/mL, and for lame horses was 5.24 ± 1.83 µg/mL (P<0.001). Antibodies against COMP neo-epitope did not stain normal articular cartilage, but intracytoplasmic staining was found in superficial chondrocytes of mild OA cartilage and in the extracellular matrix of moderately osteoarthritic cartilage. MAIN LIMITATIONS: ELISA was based on polyclonal antisera rather than a monoclonal antibody. There is a sex and breed bias within the groups of horses, also it could have been of value to include horses with septic arthritis and tendonitis and investigated joint differences. CONCLUSIONS: This COMP neo-epitope can be measured in sera, and results indicate that it could be a biomarker for pathologic fragmentation of cartilage in connection with acute joint lameness.

Cartilage oligomeric matrix protein neoepitope in the synovial fluid of horses with acute lameness: A new biomarker for the early stages of osteoarthritis.

BACKGROUND: Clinical tools to diagnose the early changes of osteoarthritis (OA) that occur in the articular cartilage are lacking. OBJECTIVES: We sought to identify and quantify a novel cartilage oligomeric matrix protein (COMP) neoepitope in the synovial fluid from the joints of healthy horses and those with different stages of OA. STUDY DESIGN: In vitro quantitative proteomics and assay development with application in synovial fluids samples obtained from biobanks of well-characterised horses. METHODS: Articular cartilage explants were incubated with or without interleukin-1ß for 25 days. Media were analysed via quantitative proteomics. Synovial fluid was obtained from either normal joints (n = 15) or joints causing lameness (n = 17) or with structural OA lesions (n = 7) and analysed for concentrations of the COMP neoepitope using a custom-developed inhibition enzyme-linked immunosorbent assay (ELISA). Explants were immunostained with polyclonal antibodies against COMP and the COMP neoepitopes. RESULTS: Semitryptic COMP peptides were identified and quantified in cell culture media from cartilage explants. A rabbit polyclonal antibody was raised against the neoepitope of the N-terminal portion of one COMP fragment (sequence SGPTHEGVC). An inhibition ELISA was developed to quantify the COMP neoepitope in synovial fluid. The mean concentration of the COMP neoepitope significantly increased in the synovial fluid from the joints responsible for acute lameness compared with normal joints and the joints of chronically lame horses and in joints with chronic structural OA. Immunolabelling for the COMP neoepitope revealed a pericellular staining in the interleukin-1ß-stimulated explants. MAIN LIMITATIONS: The ELISA is based on polyclonal antisera rather than a monoclonal antibody. CONCLUSIONS: The increase in the COMP neoepitope in the synovial fluid from horses with acute lameness suggests that this neoepitope has the potential to be a unique candidate biomarker for the early molecular changes in articular cartilage associated with OA.

Characterisation of lubricin in synovial fluid from horses with osteoarthritis.

REASON FOR PERFORMING STUDY: The glycoprotein lubricin contributes to the boundary lubrication of the articular cartilage surface. The early events of osteoarthritis involve the superficial layer where lubricin is synthesised. OBJECTIVES: To characterise the glycosylation profile of lubricin in synovial fluid from horses with osteoarthritis and study secretion and degradation of lubricin in an in vitro inflammation cartilage model. STUDY DESIGN: In vitro study. METHODS: Synovial fluid samples collected from horses with joints with normal articular cartilage and structural osteoarthritic lesions; with and without osteochondral fragments, were analysed for the lubricin glycosylation profiles. Articular cartilage explants were stimulated with or without interleukin-1ß for 25 days. Media samples collected at 3-day intervals were analysed by quantitative proteomics, western blot and enzyme-linked immunosorbent assay. RESULTS: O-glycosylation profiles in synovial fluid revealed both Core 1 and 2 O-glycans, with Core 1 O-glycans predominating. Synovial fluid from normal joints (49.5 ± 1.9%) contained significantly lower amounts of monosialylated Core 1 O-glycans compared with joints with osteoarthritis (53.8 ± 7.8%, P = 0.03) or joints with osteochondral fragments (57.3 ± 8.8%, P = 0.001). Additionally, synovial fluid from normal joints (26.7 ± 6.7%) showed higher amounts of disialylated Core 1 O-glycan than from joints with osteochondral fragments (21.2 ± 4.9%, P = 0.03). A C-terminal proteolytic cleavage site in lubricin was found in synovial fluid from normal and osteochondral fragment joints and in media from interleukin-1ß stimulated and unstimulated articular cartilage explants. CONCLUSIONS: This is the first demonstration of a change in the glycosylation profile of lubricin in synovial fluid from diseased equine joints compared with that from normal joints. We demonstrate an identical proteolytic cleavage site of lubricin both in vitro and in vivo. The reduced sialation of lubricin in synovial fluid from diseased joints may affect the boundary lubricating ability of the superficial layer of articular cartilage and could be one of the early events in the progression of osteoarthritis.

Clinical proteomics in neurodegenerative disorders.

Neurodegenerative disorders are characterized by neuronal impairment that eventually leads to neuronal death. In spite of the brain's known capacity for regeneration, lost neurons are difficult to replace. Therefore, drugs aimed at inhibiting neurodegenerative processes are likely to be most effective if the treatment is initiated as early as possible. However, clinical manifestations in early disease stages are often numerous, subtle and difficult to diagnose. This is where biomarkers that specifically reflect onset of pathology, directly or indirectly, may have a profound impact on diagnosis making in the future. A triplet of biomarkers for Alzheimer's disease (AD), total and hyperphosphorylated tau and the 42 amino acid isoform of beta-amyloid, has already been established for early detection of AD before the onset of dementia. However, more biomarkers are needed both for AD and for other neurodegenerative disorders, such as Parkinson's disease, frontotemporal dementia and amyotrophic lateral sclerosis. This review provides an update on recent advances in clinical neuroproteomics, a biomarker discovery field that has expanded immensely during the last decade, and gives an overview of the most commonly used techniques and the major clinically relevant findings these techniques have lead to.

Amyloid beta1-40 quantification in CSF: comparison between chromatographic and immunochemical methods.

BACKGROUND/AIMS: Amyloid beta (Abeta) is the principal component of senile plaques, one of the hallmarks of Alzheimer's disease (AD). Evidence is accumulating that soluble aggregates (oligomers) of Abeta are important in the pathogenesis of AD. METHODS: We compared three different methods for quantification of the 40 amino acid form of Abeta (Abeta40) in CSF, two based on antibodies [ELISA and surface-enhanced laser desorption/ionization-time of flight (SELDI-TOF) with antibody-coated arrays] and one based on direct binding of proteins to a protein array [SELDI-TOF and immobilized metal affinity [copper] (IMAC30)]. RESULTS: CSF Abeta40 concentration was only found to be significantly elevated in AD (127% of control levels; p=0.0095) using SELDI-TOF with IMAC30 arrays. CONCLUSIONS: These data suggest that the measured Abeta level in CSF may differ depending on whether antibody-based methods are used or not, possibly caused by epitope masking due to Abeta oligomerization or to binding of Abeta to carrier proteins.

Mutations in the 4-hydroxyphenylpyruvate dioxygenase gene (HPD) in patients with tyrosinemia type III.

Tyrosinemia type III (OMIM 276710) is an autosomal recessive disorder caused by the deficiency of 4-hydroxyphenylpyruvate dioxygenase (HPD), the second enzyme in the tyrosine catabolic pathway. The enzyme deficiency results in an accumulation and increased excretion of tyrosine and phenolic metabolites. Only a few cases with the disorder have been described, and the clinical spectrum of the disorder is unknown. Reported patients have presented with mental retardation or neurological symptoms or have been picked up by neonatal screening. We have identified four presumed pathogenic mutations (two missense and two nonsense mutations) in the HPD gene in three unrelated families encompassing four homozygous individuals and one compound heterozygous individual with tyrosinemia type III. Furthermore, a number of polymorphic mutations have been identified in the HPD gene. No correlation of the severity of the mutation and enzyme deficiency and mental function has been found; neither do the recorded tyrosine levels correlate with the clinical phenotype.

Human 4-hydroxyphenylpyruvate dioxygenase gene (HPD).

Overlapping DNA fragments spanning approximately 21 kb of genomic DNA and encompassing the human 4-hydroxyphenylpyruvate dioxygenase gene (HPD) have been cloned by screening a human leukocyte genomic library and by PCR amplification of human fibroblastic DNA. A continuous gene sequence of 20,890 nucleotides was established, including 1957 bp of the 5'-flanking region. The 4-hydroxyphenylpyruvate dioxygenase gene is composed of 14 exons interrupted by 13 introns, all exhibiting conventional vertebrate splicing. Computer analysis of the DNA sequence revealed 12 complete repetitive Alu elements, 1 in the 5'-flanking region and 11 in the intervening segments of the gene. The transcriptional initiation site was mapped to a position 35 nt upstream of the translational start point. The computer analysis also identified several potential transcription regulatory elements, including one CRE site, two AP-2 sites, and two Sp1 sites, in the sequence upstream of the transcription initiation site. Functional analysis of promoter activity by transient transfection of chloramphenicolacetyl transferase reporter plasmids revealed a possible involvement of cyclic adenosine monophosphate in the regulation of transcription. The highest level of expression of 4-hydroxyphenylpyruvate dioxygenase was found in human liver tissue as demonstrated by Northern blot analysis.

Alternative splicing determines the binding of platelet-derived growth factor (PDGF-AA) to glycosaminoglycans.

We have shown previously that the platelet-derived growth factor (PDGF) and a synthetic oligopeptide, corresponding to the basic carboxyl-terminal amino acid extension of the long PDGF-A isoform, bind to heparin. Here, we have expressed the long (rA125) and the short (rA109) variants of PDGF A-chains in Escherichia coli and produced the functional homodimers. Surface plasmon resonance analyses showed that while the dimeric rA125 bound with high affinity to low molecular weight heparin, the rA109, lacking the basic extension, did not. This strongly indicated that high affinity binding is due to the carboxyl-terminal extension. Investigations of kinetics and thermodynamics suggested an allosteric binding mechanism. Thus, dimeric rA125 contains two equivalent binding sites. Following low affinity binding of heparin to one binding site, the dimer undergoes a conformational change, increasing the affinity for heparin about 40 times. This positive cooperativity requires the basic amino acid extension in both monomers of the dimeric PDGF molecule. Thermodynamics of the reaction, showing an entropy-driven endothermic process, suggest the involvement of hydrophobic interactions in this rearrangement. Three amino acids in the basic carboxyl-terminal extension were essential for the interaction: the basic residues Arg111 and Lys116, and the polar Thr125. We also found that other glycosaminoglycan species, corresponding to those produced by human arterial smooth muscle cells, bound to dimeric rA125 and that heparan sulfate showed the highest affinity.

Expression of the leukocyte-associated sialoglycoprotein CD43 by a colon carcinoma cell line.

The colon adenocarcinoma cell line COLO 205 secretes L-CanAg, a mucin-like glycoprotein carrying the carcinoma-associated sialyl-Lewis a carbohydrate epitope. In an attempt to identify its apoprotein, an NH2-terminal peptide sequence was obtained from purified L-CanAg. In all interpretable positions, this sequence showed 100% identity to the NH2-terminal of human CD43 (leukosialin, sialophorin), a plasma membrane-bound sialoglycoprotein hitherto only identified in leukocytes and other hematopoietic cells. An antiserum against deglycosylated L-CanAg and an anti-CD43 antiserum both immunoprecipitated a 61-kDa band, interpreted as the CD43 precursor, from COLO 205 cells as well as from the known CD43-expressing cell line HL-60. Results from immunoprecipitations following pulse-chase experiments and tunicamycin treatments were in agreement with earlier studies on the CD43 precursor. RNA blot analysis confirmed the expression of CD43 by the COLO 205 cell line, whereas three other colon carcinoma cell lines were negative. The glycosylation-dependent monoclonal antibody Leu-22, which recognizes leukocyte CD43, failed to bind L-CanAg, probably due to its much more extensive glycosylation. We conclude that L-CanAg is the secreted extracellular domain of a novel glycoform of CD43 and that CD43, if expressed in other carcinoma cells, may have escaped notice in studies relying on glycosylation-dependent monoclonal antibodies against leukocyte CD43.

Regional assignment of the human 4-hydroxyphenylpyruvate dioxygenase gene (HPD) to 12q24-->qter by fluorescence in situ hybridization.

Using a panel of human-rodent somatic cell hybrids, we have previously mapped the gene (HPD, previously called PPD) encoding 4-hydroxyphenylpyruvate dioxygenase to the distal half of the long arm of human chromosome 12, region q14-->qter. To obtain a genomic probe useful for fluorescence in situ hybridization (FISH) analysis we screened a human leukocyte genomic library and isolated a 13.4-kb phage clone, which by restriction fragment and sequence analyses was shown to contain exons 1-10 of HPD and approximately 2-kb upstream sequences. We now report the subregional localization of HPD to 12q24-->qter based on two color FISH analysis employing this clone.

Human 4-hydroxyphenylpyruvate dioxygenase. Primary structure and chromosomal localization of the gene.

We report the primary structure of 4-hydroxyphenylpyruvate dioxygenase [4-hydroxyphenyl-pyruvate:oxygen oxidoreductase (hydroxylating, decarboxylating)]. The work is based on the isolation of cDNA clones from human liver lambda gt11 libraries. Several overlapping clones covering the coding sequence were characterized. In parallel, peptides from four different digests of the purified protein were analysed for their amino-acid sequence. These peptide sequences covered 86% of the cDNA-derived amino-acid sequence. This gives the sequence for a polypeptide of 392 amino acids with a calculated molecular mass of 44.8 kDa. There is more than 80% identity between the human and the pig enzymes and also between these enzymes and the F antigen from rat and the two allelic forms of this antigen from mouse. The enzyme has 53% conserved amino acids and 27% identical amino acids in common with 4-hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. P.J. 874 and 52% conserved and 28% identical residues, with a protein from Shewanella colwelliana. At the C-terminus there is 61% identity between the seven proteins. These results indicate that these proteins are all 4-hydroxyphenylpyruvate dioxygenases. The identity of the C-terminus makes this part of the molecule a candidate for a functional role in the catalytic process. At conserved positions in all seven enzymes, there are two tyrosine residues and three histidine residues, i.e. amino acids which have been implicated as ligands for iron in 2-oxoacid-dependent dioxygenases. The gene encoding the enzyme was localized to chromosome 12q14-->qter by Southern-blot analysis of human-rodent somatic-cell hybrids.

gamma-Butyrobetaine hydroxylase. Structural characterization of the Pseudomonas enzyme.

gamma-Butyrobetaine hydroxylase is a 2-oxoglutarate-dependent dioxygenase that catalyzes the hydroxylation of gamma-butyrobetaine to carnitine, the last step in the biosynthesis of carnitine from lysine. The primary structure of the enzyme from Pseudomonas sp. AK1 has been determined. Sequence analysis of the intact protein and of peptides from essentially three different digests established the presence of a peptide chain containing 383 residues, and an N-terminal truncated form of 382 residues. The two chains have molecular masses of 43,321 Da and 43,207 Da, respectively, and are identical except for the presence or absence of an N-terminal asparagine residue; the shorter form starts with an alanine residue. In preparations of the dimeric protein, the two chains occur in an approximate ratio of 1:1. There are nine cysteine residues and 13 histidine residues, i.e. amino acids which have been postulated as ligands for iron binding. In spite of functional similarities, there appears to be no clear sequence similarities with any of the other mammalian 2-oxoglutarate-dependent dioxygenases so far characterized.

Characterization of 4-hydroxyphenylpyruvate dioxygenase. Primary structure of the Pseudomonas enzyme.

The primary structure of Pseudomonas 4-hydroxyphenylpyruvate dioxygenase was determined. Sequence degradation of the intact protein and of peptides from three different digests of the carboxymethylated protein established a 357-residue polypeptide chain with a free alpha-amino group. Hydroxylamine cleavage at a single Asn-Gly sequence was useful. Comparisons with known structures in data banks revealed no close relationship with other characterized proteins. The human enzyme has a related composition, suggesting that also the eukaryotic form belongs to this protein type, but with a blocked N-terminus like in many other eukaryotic intracellular proteins. Secondary structure predictions suggest an alpha/beta mixed structure, fairly typical of globular proteins, without long segments of hydrophobicity or charge, although a region in the middle of the C-terminal third of the subunit appears to have the most extreme properties. A ferric centre, correlating with enzyme activity and absorbance at 595 nm, has previously been assigned to tyrosinate coordination. The Tyr and His distributions, and the position of a single Cys residue, all suggest a few likely sites, outside the C-terminal segment, for this centre.