I-cell disease and pseudo-Hurler polydystrophy result from a deficiency of GlcNAc-1-phosphate transferase (GlcNAcPTase) which is involved in the formation of mannose 6-phosphate on lysosomal enzymes for their targeting to lysosomes. Although a single enzyme defect is responsible for these disorders our biochemical and genetic studies have demonstrated the existence of several complementation groups indicating that more than one gene mutation affects the expression of GlcNAcPTase. A major goal of this laboratory is to resolve the molecular basis for each complementation group. An integral part of these studies requires the production of polyclonal antiserum to the purified enzyme or its synthetic peptides. The short term goal of the current proposal is to obtain highly purified or purified GlcNAcPTase by employing the newly developed Affigel 501, uteroferrin-Avidgel Ax, and 5(3-allylamine)-UDP-GlcNAc-Sepharose 4B affinity chromatographies. Subsequent use of a unique photoaffinity labeling technique with [32P]4-SUDP in conjunction with preparative SDS- PAGE can be used to identify and isolate the catalytic subunit of the enzyme. Following amino acid sequencing of the subunit, peptides will be synthesized to segments of the sequence, and polyclonal antibodies prepared to these sequences. The resulting positive antibodies can be used to immunoaffinity purify the GlcNAcPTase. Amino acid sequencing and antipeptide antibodies can then be prepared against other domains in a similar manner as for the catalytic domain. The resulting antibodies that cross-react with GlcNAcPTase will be used to determine whether the gene mutation(s) responsible for the various complementation groups affect the biosynthesis and/or post-translational processing of the GlcNAcPTase. In addition, using these specific antibodies collaborative studies will be carried out to clone and map the gene(s) controlling the expression of GlcNAcPTase.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS012138-17
Application #
2262392
Study Section
Medical Biochemistry Study Section (MEDB)
Project Start
1978-06-01
Project End
1996-06-30
Budget Start
1994-07-01
Budget End
1995-06-30
Support Year
17
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Type
Schools of Medicine
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093
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Miller, A L; Norton, V; Robertson, R et al. (1993) Light and heavy lysosomes: characterization of N-acetyl-beta-D-hexosaminidase isolated from normal and I-cell disease lymphoblasts. Glycobiology 3:313-8
DiCioccio, R A; Miller, A L (1993) Phosphorylation and subcellular location of alpha-L-fucosidase in lymphoid cells from patients with I-cell disease and pseudo-Hurler polydystrophy. Glycobiology 3:489-95
Zhao, K W; Yeh, R; Miller, A L (1992) Purification and characterization of human lymphoblast N-acetylglucosamine-1-phosphotransferase. Glycobiology 2:119-25
Dicioccio, R A; Miller, A L (1992) Binding receptors for alpha-L-fucosidase in human B-lymphoid cell lines. Glycoconj J 9:56-62
DiCioccio, R A; Miller, A L (1991) Biosynthesis, processing, and secretion of alpha-L-fucosidase in lymphoid cells from patients with I-cell disease and pseudo-Hurler polydystrophy. Glycobiology 1:595-604
Little, L; Alcouloumre, M; Drotar, A M et al. (1987) Properties of N-acetylglucosamine 1-phosphotransferase from human lymphoblasts. Biochem J 248:151-9
Hatton, J D; Cox, G F; Miller, A L et al. (1987) Identification of polypeptides associated with sarcolemmal vesicles enriched in orthogonal arrays. Biochim Biophys Acta 904:373-80
Little, L E; Mueller, O T; Honey, N K et al. (1986) Heterogeneity of N-acetylglucosamine 1-phosphotransferase within mucolipidosis III. J Biol Chem 261:733-8