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Title:Purification and Characterization of the Drop-1 Proteoglycan in Drosophila Melanogaster
Author(s):Graner, Michael William
Doctoral Committee Chair(s):Karr, Timothy,
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Biology, Cell
Chemistry, Biochemistry
Abstract:In an effort to identify molecules involved in Drosophila fertilization, probes against these molecules were generated. Using Drosophila testis as antigen, monoclonal antibodies were isolated that recognize three high molecular weight molecules present on Drosophila sperm and in the fertilized egg. The biochemical and cell biological properties of the immunoreactive proteins were determined.
Microscopic examination of Drosophila embryos revealed that one of these antibodies is highly specific for Drosophila sperm tail epitopes, while the other four recognize not only the sperm tail but embryonic structures distributed throughout the egg in vesicular and/or punctate patterns.
Biochemical characterization of the antigens demonstrated that each of the five antibodies recognized the same set of high molecular weight ($>$200 kD) acidic (pI 3.0-3.5) proteins. Further analysis of these antigens revealed that they were proteoglycan-like glycoproteins as determined by chemical deglycosylation methods, including trifluoromethylsulfonate treatment, alkaline borohydride treatment, and nitrous acid deamination and cleavage of heparin/heparan sulfate. In each case, the antigen's behavior following chemical treatment was consistently like a proteoglycan. Further characterizations indicated that the antigens are sulfated, as is expected of a proteoglycan. This protein has been named DROP 1, for underbarDrosophila Proteoglycan 1, and is only the third individual proteoglycan characterized in Drosophila. It is present at all stages of the Drosophila life cycle, in several Drosophila cell lines, and in at least one other organism.
DROP 1 purification involved extraction of embryonic material in guanidine hydrochloride, preparative isoelectric focusing, proteolytic digestion, and immunoaffinity techniques. The purification revealed the presence of other low abundance immunoreactive proteins that may be members of a family related by a probable carbohydrate epitope.
DROP 1 antibodies also recognize other proteins in Drosophila testis tissue and sperm. N-terminal amino acid sequence analysis of a gel-purified testis protein revealed homology to Drosophila calreticulin, a calcium-binding protein found in a wide variety of organisms and cell types.
DROP 1 is thus a widely distributed family of glycoproteins/proteoglycans whose possible functions as cell-surface and extracellular matrix molecules are reflected in their heterogeneous forms and functions.
Issue Date:1993
Type:Text
Description:151 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1993.
URI:http://hdl.handle.net/2142/72362
Other Identifier(s):(UMI)AAI9411637
Date Available in IDEALS:2014-12-17
Date Deposited:1993


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