Biomedical subjects

P A McCourt

Publications and source records attributed to P A McCourt.

10 recordsLinked to original sources

PubMed1999

Characterization of a hyaluronan receptor on rat sinusoidal liver endothelial cells and its functional relationship to scavenger receptors.

Hyaluronan is a widely distributed extracellular component of connective tissue with several mechanical and cell biological functions. The serum level of hyaluronan is elevated in rheumatic and liver diseases and in certain malignancies. The major route of hyaluronan clearance from the blood is via the liver, taken up predominantly by sinusoidal liver endothelial cells. We have purified a novel hyaluronan binding protein from liver that also has an affinity for the N-terminal propeptide of type I procollagen, a physiological scavenger receptor ligand. A polyclonal antibody raised against the protein was found to inhibit the binding and degradation of hyaluronan as well as two scavenger receptor ligands by cultured sinusoidal liver endothelial cells. Immunostaining of nonpermeabilized liver cells and liver sections showed that the antibody specifically stains the surface of sinusoidal liver endothelial cells. After pretreatment with monensin to block the recirculation of endocytic receptors, the immunostaining was specifically associated with early endosomes of these cells. Thus, this rat sinusoidal liver endothelial cell hyaluronan receptor shares functional properties with the scavenger receptor family, a group of proteins shown to play a key role in the uptake of atherogenic lipids and other waste products from the tissues.

Animals↗

PubMed1999

How does the hyaluronan scrap-yard operate?

Hyaluronan is an extracellular polysaccharide found throughout the extracellular matrix, especially in soft connective tissue. It has an unusual feature, in that its turnover rate is much greater than that of other extracellular matrix components. The mechanisms of its synthesis at the plasma membrane (by hyaluronan synthases) and lysosomal degradation (by hyaluronidases) are well documented. However, the mechanisms by which it enters those cells primarily involved in its degradation remain a mystery. Recent work now suggests that a novel scavenger receptor expressed on the surface of liver endothelial cells is responsible for part of this degradative process. Further study is required to fill the remaining gaps in our knowledge about this process in other tissues.

Animals↗

PubMed1997

On the adsorption of hyaluronan and ICAM-1 to modified hydrophobic resins.

Hyaluronan is a negatively charged glycosaminoglycan that occurs in connective tissue and has a wide range of mechanical and cell biological functions. The purpose of this study was to utilize affinity chromatography resins for purification of detergent (Triton X-100) solubilized hyaluronan binding proteins from liver, the major organ of hyaluronan clearance from the blood. However, during these studies we made the unexpected finding that hyaluronan binds to Sepharose substituted with a hexamethylene chain, a commonly used spacer arm in affinity chromatography resins, capped with either a terminal primary amine or a terminal acetoamido group. Hyaluronan did not bind the hydrophobic resins hexyl- or octyl-Sepharose under the same conditions. It was also found that rat liver intercellular adhesion molecule-1 binds to resins containing the hexamethylene spacer arm, an interaction which could be inhibited with free hyaluronan oligosaccharides. Finally, we have determined that resins with ethylene spacer arms show no affinity for hyaluronan and can therefore be used to immobilize hyaluronan for chromatography of hyaluronan binding proteins. By using this resin we have purified two proteins of approximately 200 and 400 kDa from rat liver endothelial cells. In summary, this study demonstrates the efficacy of certain "capped-hydrophobic" resins for binding hyaluronan; these resins may provide a novel means for the study and/or purification of this glycosaminoglycan. This study further demonstrates the importance of the careful design of appropriate affinity columns for the specific purification of hyaluronan binding proteins.

Adsorption↗

PubMed1994

Intercellular adhesion molecule-1 is a cell surface receptor for hyaluronan.

Our laboratory has previously characterized and purified the hyaluronan receptor by hyaluronan affinity chromatography of rat liver endothelial cells. We have now isolated the receptor from whole rat liver and have obtained sufficient quantities for amino acid sequence analysis. Four peptides of various lengths were obtained from affinity-purified receptor and found to have identity with rat intercellular adhesion molecule-1. This glycoprotein is normally expressed in low amounts on the endothelial cells, but is up-regulated in inflamed and malignant tissues, and mediates cell-cell adhesion as a ligand for lymphocyte function-associated antigen-1 and the macrophage-associated Mac-1. The affinity of intercellular adhesion molecule-1 for hyaluronan is likely to have important implications for cell adhesion in normal and in disease states such as inflammation, atherosclerosis, and cancer.

Amino Acid Sequence↗

PubMed1992

Hurler syndrome: a patient with abnormally high levels of alpha-L-iduronidase protein.

Mucopolysaccharidosis type I (MPS I: McKusick 25280) is a clinically heterogenous lysosomal storage disorder which is caused by a variable deficiency in alpha-L-iduronidase activity (alpha-L-iduronide iduronohydrolase, EC 3.2.1.76). Cultured fibroblasts from an MPS I patient (cell line 2827) with a severe clinical phenotype (Hurler syndrome) have been characterized using immunochemical and biochemical techniques. Using a specific immunoquantification assay, we have demonstrated that cell line 2827 had an alpha-L-iduronidase protein content (189 ng/mg of extracted cell protein) at least six times greater than the mean level found in normal control fibroblasts (30 ng/mg of extracted cell protein). This was the only MPS I cell line, from a group of 23 MPS I patients, that contained greater than 7% of the mean level of alpha-L-iduronidase protein detected in normal controls. Cell line 2827 had very low alpha-L-iduronidase activity toward the fluorogenic substrate 4-methylumbelliferyl-alpha-L-iduronide, and a radiolabeled disaccharide substrate derived from heparin. Maturation studies of alpha-L-iduronidase in cell line 2827 showed apparently normal levels of alpha-L-iduronidase synthesis with delayed processing to the mature form. Subcellular fractionation experiments demonstrated alpha-L-iduronidase protein in lysosomal-enriched fractions isolated from cell line 2827, suggesting a normal cell distribution and supporting the proposed delayed processing. It is proposed that the MPS I patient described has an alpha-L-iduronidase gene mutation which affects both the active site and post-translational processing of the enzyme. This mutation must be structurally conservative because it does not result in instability either during maturation or in the lysosome.

Cells, Cultured↗

PubMed1992

Immunoquantification and enzyme kinetics of alpha-L-iduronidase in cultured fibroblasts from normal controls and mucopolysaccharidosis type I patients.

alpha-L-Iduronidase activity is deficient in mucopolysaccharidosis type I (MPS I; Hurler syndrome, Scheie syndrome) patients and results in the disruption of the sequential degradation of the glycosaminoglycans dermatan sulfate and heparan sulfate. A monoclonal antibody-based immunoquantification assay has been developed for alpha-L-iduronidase, which enables the detection of at least 16 pg alpha-L-iduronidase protein. Cultured human skin fibroblasts from 12 normal controls contained 17-54 ng alpha-L-iduronidase protein/mg extracted cell protein. Fibroblasts from 23 MPS I patients were assayed for alpha-L-iduronidase protein content. Fibroblast extracts from one MPS I patient contained at least six times the level of alpha-L-iduronidase protein for normal controls--but contained no associated enzyme activity--and is proposed to represent a mutation affecting the active site of the enzyme. Fibroblast extracts from 11 MPS I patients contained 0.05-2.03 ng alpha-L-iduronidase protein/mg extracted cell protein, whereas immunodetectable protein could not be detected in the other 11 patients. Four fibroblast extracts with no immunodetectable alpha-L-iduronidase protein had residual alpha-L-iduronidase activity, suggesting that the mutant alpha-L-iduronidase in cultured cells from these MPS I patients has been modified to mask or remove the epitopes detected by two monoclonal antibodies used in the quantification assay. Both the absence of immunoreactivity in a mild MPS I patient and high protein level in a severe MPS I patient present limitations to the use of immunoquantification analysis as a sole measure of patient phenotype. Enzyme kinetic analysis of alpha-L-iduronidase from MPS I fibroblasts revealed a number of patients with either abnormal substrate binding or catalytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗

PubMed1991

A specific fluorogenic assay for N-acetylgalactosamine-4-sulphatase activity using immunoadsorption.

A method combining immune capture and enzyme detection by fluorochemistry has been developed for the diagnostic assay of N-acetylgalactosamine-4-sulphatase (4-sulphatase). The procedure uses a monoclonal antibody 4-S 4.1 to immunoadsorb 4-sulphatase specifically from complex protein samples containing other sulphatases, and 4-methylumbelliferyl sulphate to detect captured 4-sulphatase. The assay provides an accurate and simple method for the diagnosis of Maroteaux-Lamy syndrome (Mucopolysaccharidosis type VI).

Antibodies, Monoclonal↗

PubMed1991

Analysis of N-acetylgalactosamine-4-sulfatase protein and kinetics in mucopolysaccharidosis type VI patients.

A sensitive and specific, monoclonal antibody-based immunoquantification assay has facilitated determination of the N-acetylgalactosamine-4-sulfatase (4-sulfatase) protein content in cultured fibroblasts from normal controls and mucopolysaccharidosis type VI (MPS VI) patients. The assay enabled the quantification of 4-sulfatase protein by using a panel of seven monoclonal antibodies and has shown that fibroblasts from 16 MPS VI patients contained less than or equal to 5% of the level determined for normal controls. Fibroblasts from the most severely affected patients contained the lowest levels of 4-sulfatase protein, usually with few epitopes detected, while fibroblasts from mildly affected patients had higher levels of 4-sulfatase protein, with all seven epitopes detected. The pattern of epitope expression is proposed to reflect the conformational changes in the 4-sulfatase protein that arise from different mutations in the 4-sulfatase gene. Immunoquantification in combination with a specific and highly sensitive 4-sulfated trisaccharide-based assay of enzyme activity in these MPS VI patient fibroblasts enabled the determination of residual 4-sulfatase catalytic efficiency (kcat/Km). The capacity of fibroblasts to degrade substrate (catalytic capacity) was calculated as the product of 4-sulfatase catalytic efficiency and the content of 4-sulfatase in fibroblasts. One patient, 2357, with no clinical signs of MPS VI but with reduced 4-sulfatase activity and protein (both 5% of normal) and dermatansulfaturia, had 5% of normal catalytic capacity. The other 15 MPS VI patient fibroblasts had 0%-1.4% of the catalytic capacity of fibroblasts from normal controls and were representative of the spectrum of MPS VI clinical phenotypes, from severe to mild.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗

PubMed1990

Immunoquantification of the low abundance lysosomal enzyme N-acetylgalactosamine 4-sulphatase.

The low abundance lysosomal enzyme N-acetylgalactosamine 4-sulphatase (4-sulphatase) has been quantified using a microimmunopurification step and a monoclonal-based ELISA detection system. The assay is similar in principle to a two-site ELISA but uses a single monoclonal antibody against one epitope to bind 4-sulphatase in two separate assay steps. The sensitivity of this assay is sufficient to allow the quantification of 4-sulphatase in human cultured skin fibroblasts derived from normal controls and patients deficient in 4-sulphatase activity (mucopolysaccharidosis type VI or Maroteaux-Lamy syndrome). The results obtained suggest a range of mucopolysaccharidosis type VI or 4-sulphatase deficient mutants, from those expressing little or no quantifiable 4-sulphatase protein to those examples with quantifiable levels of 4-sulphatase protein which is enzymically inactive. Phenotypic variability in patients with a 4-sulphatase deficiency may therefore be partially attributed to a range of protein expressions. The method should allow the determination of 4-sulphatase specific activity in mucopolysaccharidosis type VI patients.

Antibodies, Monoclonal↗

PubMed1989

Immunopurification and characterization of human alpha-L-iduronidase with the use of monoclonal antibodies.

alpha-L-Iduronidase from human liver was purified by a three-step five-column procedure and by immunoaffinity chromatography with a monoclonal antibody raised against purified enzyme. Seven bands identified by staining with Coomassie Blue had molecular masses of 74, 65, 60, 49, 44, 18 and 13 kDa and were present in both preparations of the liver enzyme. However, relative to the immunopurification procedure, alpha-L-iduronidase purified by the five-column procedure was considerably enriched in the 65 kDa polypeptide band. The seven bands were identified by Western-blot analysis with two different monoclonal antibodies raised against alpha-L-iduronidase. The chromatographic behaviour of alpha-L-iduronidase on the antibody column was dependent upon the quantity of enzyme loaded. Above a particular load concentration a single peak of enzyme activity was eluted, whereas at load concentrations below the critical value alpha-L-iduronidase was eluted in two peaks of activity, designated form I (eluted first) and form II (eluted second). The following properties of the two forms of alpha-L-iduronidase were determined. (1) The two forms from liver were composed of different proportions of the same seven polypeptides. (2) When individually rechromatographed on the antibody column, each form from liver shifted to a more retarded elution position but essentially retained its chromatographic behaviour relative to the other form. (3) Forms I and II of liver alpha-L-iduronidase showed no difference in their activities towards disaccharide substrates derived from two glycosaminoglycan sources, heparan sulphate and dermatan sulphate. (4) The native molecular size of forms I and II of liver alpha-L-iduronidase was 65 kDa as determined by gel-permeation chromatography. (5) Immunoaffinity chromatography of extracts of human lung and kidney resulted in the separation of alpha-L-iduronidase into two forms, each with different proportions of the seven common polypeptide species. (6) Lung forms I and II were taken up readily into cultured skin fibroblasts taken from a patient with alpha-L-iduronidase deficiency. Liver forms I and II were not taken up to any significant extent. Lung form II gave intracellular contents of alpha-L-iduronidase that were more than double those of normal control fibroblasts, whereas lung form I gave contents approximately equal to normal control values. We propose that all seven polypeptides are derived from a single alpha-L-iduronidase gene product, and that different proportions of these polypeptides can function as a single alpha-L-iduronidase entity.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗