Molecular genetic studies of human and feline N-acetylgalactosamine 4-sulfatase (arylsulfatase B).
Item
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Title
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Molecular genetic studies of human and feline N-acetylgalactosamine 4-sulfatase (arylsulfatase B).
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Identifier
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AAI9224824
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identifier
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9224824
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Creator
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Jackson, Christine Elise.
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Contributor
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Adviser: Edward H. Schuchman
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Date
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1992
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Genetics | Biology, Molecular
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Abstract
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N-acetylgalactosamine 4-sulfatase (Arylsulfatase B; ASB; EC 3.1.6.1) is the lysosomal enzyme responsible for the hydrolysis of 4-sulfate groups from N-acetylgalactosamine 4-sulfate moieties present in the glycosaminoglycan, dermatan sulfate. A deficiency of this enzyme in man results in the lysosomal storage disorder, Maroteaux-Lamy disease (Mucopolysaccharidosis Type VI; MPS VI). Notably, a naturally occurring model of MPS VI has been identified in Siamese cats, and the biochemical and pathological properties of the human and feline diseases have been well characterized.;Thus, the molecular genetic studies described in this thesis were undertaken in order to provide insights into the nature of this lysosomal hydrolase, and to facilitate the future use of this prototypic animal model system for the development of enzyme and/or gene replacement therapy for MPS VI. Human ASB was purified from liver and five tryptic peptides (112 residues) were microsequenced. Degenerative oligonucleotide probes were constructed and used to isolate overlapping partial cDNAs for ASB by the mixed oligonucleotide-primed amplification of cDNA (MOPAC) cloning strategy. The complete human ASB cDNA sequence is 2,802 bp and includes 559 bp of 5{dollar}\sp\prime{dollar} untranslated sequence, 644 bp of 3{dollar}\sp\prime{dollar} untranslated sequence, and a 1,599 bp open reading frame encoding 533 amino acids. There are six potential N-glycosylation sites. Two full-length cDNAs, differing only in the length of their 5{dollar}\sp\prime{dollar} untranslated sequences, were constructed from the partial cDNAs, and transiently expressed in COS-1 cells to demonstrate their functional integrity. These cDNAs have also been subcloned into the retroviral vectors, DCTK and pBC140, so that gene transfer studies may be initiated.;Feline ASB cDNAs have also been isolated using a 2.2 kb human ASB cDNA as a probe. The feline cDNA sequence, compiled from the overlapping cDNA clones, is 1,939 bp and includes 3 bp of 5{dollar}\sp\prime{dollar} untranslated sequence, 331 bp of 3{dollar}\sp\prime{dollar} untranslated sequence, and 1,605 bp coding for 535 amino acids. There are five potential N-glycosylation sites. A full-length feline cDNA was constructed and transiently expressed to demonstrate its functional integrity. This feline cDNA is now available for retroviral studies, if necessary. Additionally, the feline ASB gene was mapped to feline chromosome A1 by PCR analysis of somatic cell hybrid panels.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
