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Biomedical subjects

W S Sly

Publications and source records attributed to W S Sly.

At least 19 recordsLinked to original sources
PubMed1979

Enzymatic identification of mannose 6-phosphate on the recognition marker for receptor-mediated pinocytosis of beta-glucuronidase by human fibroblasts.

Human beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31), like many other glycoprotein lysosomal hydrolases, is subject to receptor-mediated endocytosis by fibroblasts. Prior work demonstrated charge heterogeneity in beta-glucuronidase and showed that high-uptake forms are more acidic than slowly internalized forms. Considerable indirect evidence implicated mannose 6-phosphate as an essential part of the recognition marker on high-uptake enzyme forms. Here we report the purification of beta-glucuronidase from human spleen and demonstrate enzymatically that mannose 6-phosphate is released on acid hydrolysis of pure enzyme varies directly with its susceptibility to pinocytosis by fibroblasts. Enzyme forms resolved by CM-Sephadex chromatography differed over an 18-fold range in uptake rate and in mannose 6-phosphate content. The most acidic forms had 4.4 mol of mannose 6-phosphate per mol of enzyme. The mannose 6-phosphate was released from the enzyme by treatment with endoglycosidase H with concomitant loss of susceptibility to adsorptive endocytosis. Thus, these studies provide direct evidence that mannose 6-phosphate is present on high-uptake enzyme forms, that it is present in the recognition marker for uptake, and that it is present on oligosaccharide that is released by endoglycosidase H.

Biological Transport, Active↗
PubMed1978

Sanfilippo A syndrome in the fetus.

A family is reported in which Sanfilippo A syndrome affected three siblings: the proband and twin premature infants. The feasibility of intrauterine diagnosis of mucopolysaccharidoses (MPS) Type IIIA, was demonstrated by the excessive accumulation of 35SO4-mucopolysaccharides in fibroblasts cultured from amniotic fluid obtained by amniocentesis Cross-correction studies and enzymatic analysis of cultured skin fibroblasts from the proband and the infants revealed the absence of the MPS IIIA correction factor, heparan sulfate sulfatase. However, when the premature infants expired shortly after birth, no central nervous system histopathology or ultrastructural abnormalities were found. From these observations it would appear the the third trimester fetus with MPS type IIIA has little CNS involvement.

Amniocentesis↗
PubMed1978

Receptor-mediated uptake of lysosomal enzymes.

This paper reviews the evidence for two different forms of carbohydrate-mediated recognition of lysosomal enzymes by cell surface receptors. The recognition marker on lysosomal enzymes which are rapidly pinocytosed by fibroblasts contains phosphate, probably linked to D-mannose as a phosphomannosyl moiety. The fraction of lysosomal hydrolases bearing this recognition marker for fibroblasts varies, depending on the tissue source. Another form of recognition accounts for rapid plasma clearance of infused lysosomal enzymes in the rat. This rapid clearance is mediated by receptors on Kupffer cells and other reticuloendothelial cells that recognize mannosyl residues on the glycoprotein hydrolases.

Animals↗
PubMed1977

Phosphohexosyl components of a lysosomal enzyme are recognized by pinocytosis receptors on human fibroblasts.

Human beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31), like many other glycoprotein lysosomal hydrolases, is specifically taken up from the culture medium by human fibroblasts. Prior work has indicated that the enzyme exhibits charge heterogeneity and that "high-uptake" forms, i.e., those rapidly internalized by human fibroblasts, are more acidic than slowly internalized forms. Here we present two lines of evidence that the acidic group required for the high-uptake property of certain forms of the enzyme is a phosphate on, or in proximity to, a D-mannose-type carbohydrate. The first line of evidence was obtained from analysis of inhibition of enzyme pinocytosis by yeast mannans, phosphorylated sugars, and sugars. Mannans that contained phosphate were more potent inhibitors than those that did not contain phosphate. D-Mannose 6-phosphate was a more potent inhibitor than either D-mannose 1 phosphate or 2-deoxy-D-glucose 6-phosphate. D-Mannose and certain related sugars were weak pinocytosis inhibitors, while 2- and 4-epimers of mannose were noninhibitory. Competitive inhibition was demonstrated and the apparent Kis estimated for the following compounds: Saccharomyces cerevisiae mannan from mutant X2180-mnnl, 3 X 10(-6) M; mannan from wild-type S. cerebisiae, 3 X 10(-5) M; D-mannose 6-phosphate, 6 X 10(-5) M; L-fucose, 4 X 10(-2) M; and D-mannose, 6 X 10(-2) M. The second line of evidence comes from the observation that alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] treatment of human platelet beta-glucuronidase abolished its "high-uptake" activity, without diminishing its catalytic activity, and converted some forms of the heterogeneous enzyme to less acidic forms.

Alkaline Phosphatase↗
PubMed1977

Human beta-glucuronidase deficiency mucopolysaccharidosis: identification of cross-reactive antigen in cultured fibroblasts of deficient patients by enzyme immunoassay.

An enzyme immunoassay for human beta-glucuronidase was developed to determine the presence or absence of antigenically cross-reactive material (CRM) in patients with beta-glucuronidase deficiency mucopolysaccharidosis. This assay provided a sensitive means of measuring the primary interaction between the enzyme molecule and antibody but required neither pure antigen nor monospecific antiserum, an important consideration, since neither of these was available. Goat antiserum to partially purified human placenta beta-glucuronidase did not recognize differences in normal enzyme from human placenta, liver, fibroblasts, or blood platelets. CRM was identified in fibroblast extracts from all four of the unrelated beta-glucuronidase-deficient patients studied, but titration patterns indicated genetic heterogeneity among these four mutant proteins. Fibroblast enzymes from two obligate heterozygotes were distinguishable immunologically from normal enzyme. The enzyme immunoassay was also used to compare human enzyme with liver enzyme from other mammalian species. CRM was present in liver extracts of all species tested, but the liver enzymes, except for the rabbit, were weakly cross-reactive. We conclude that despite certain limitations, the enzyme immunoassay for human beta-glucuronidase is useful and that all four beta-glucuronidase-deficient patients studied possess CRM.

Antigens↗
PubMed1977

Human beta-glucuronidase. I. Recognition and uptake by animal fibroblasts suggests animal models for enzyme replacement studies.

As part of an effort to develop an animal model for studies of uptake of human beta-glucuronidase, fibroblasts were established from primary explants of connective tissue from nine different animal species, and examined for their ability to take up human platelet beta-glucuronidase. Endogenous fibroblast beta-glucuronidase was inactivated by heating extracts to 65 degrees for 30 min. Human beta-glucuronidase was stable to this treatment. Uptake of human beta-glucuronidase by animal fibroblasts was measured as heat-stable beta-glucuronidase present in fibroblasts after exposure to partially purified human platelet beta-glucuronidase for 48 hr. Althought all animal fibroblasts examined exhibited some uptake capacity for human beta-glucuronidase, the uptake capacity of different animal fibroblasts varied over a 10-fold range. The uptake capacity of bovine fibroblasts was at least 80% that of human fibroblasts. Rat and hamster fibroblasts showed about half the uptake capacity of human fibroblasts. The rat fibroblasts resembled the human fibroblasts in the kinetics of uptake of hihg uptake (platelet) enzyme, poor uptake of human placental enzyme, and lack of appreciable turnover of enzyme taken up over 4 days. Heating extracts of rat organs containing added human beta-glucuronidase at 65 degrees selectively inactivated rat enzyme.

Animals↗
PubMed1976

Enveloped virus acquires membrane defect when passaged in fibroblasts from I-cell disease patients.

Sindbis virus obtained after passage on human fibroblasts from patients with I-cell disease (mucolipidosis II) and called I-cell virus differed from Sindbis virus obtained from chick fibroblasts or from normal human fibroblasts in two ways: (1) The I-cell virus was extremely unstable to freezing and thawing, (2) The I-cell virus showed greatly exaggerated sensitivity to inactivation by Triton X-100. Sindbis virus from fibroblasts from two patients with mucolipidosis III, a milder form of I-cell disease, showed similar, though milder, freeze-sensitivity. When freeze-sensitive I-cell virus was passaged once in mouse L-cells or normal human fibroblasts, the virus was no longer abnormal. The viral glycoproteins of I-cell virus were not distinguishable from viral glycoproteins of controls by sodium dodecyl sulfate gel electrophoresis. Gel filtration of the glycopeptides suggested small differences in two of the four glycopeptides. These findings indicate that Sindbis virus is phenotypically altered when grown on I-cell fibroblasts. These alterations must be attributed to viral envelope components derived from the host plasma membrane (membrane lipids) or to alterations in viral envelope glycoproteins. In either case, the alterations appear related to the genetic defect in I-cell fibroblasts. From these results it is clear that enveloped viruses can be useful to demonstrate and to analyze membrane defects in certain human diseases. The phenotypically altered viruses may, in turn, provide probes for studying the functional relationships of virus membrane components.

Cell Membrane↗
PubMed1976

Permanent lymphoid lines from genetically marked lymphocytes: success with lymphocytes recovered from frozen storage.

Permanent human lymphoid cell lines were established successfully from peripheral blood lymphocytes which had been separated for HL-A typing and stored in liquid nitrogen for two years. Frozen lymphocytes were chosen from two siblings who were homozygous at the LA and FOUR HL-A loci. Thawed lymphocytes were transformed with EB virus produced by the marmoset lymphoid line B95-8. No chromosome abnormalities were seen on karyotypes prepared on cells from the established human lymphoid lines using G and Q banding techniques. HL-A typing showed the expected HL-A antigens plus a considerable number of additional reactions. Separation of lymphocytes and freezing them for possible future use requires a relatively small investment. This method of preserving cells can be applied to patients with interesting genetic disorders or other biochemical markers to provide cells which can be transformed and propagated years later.

Blood Preservation↗