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Date of Award

2006

Document Type

Dissertation - Pacific Access Restricted

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Chemistry

First Advisor

Andreas H. Franz

First Committee Member

Patrick R. Jones

Second Committee Member

Jianhua Ren

Third Committee Member

Elizabeth Day

Fourth Committee Member

Craig A. Vierra

Abstract

The disulfide bond pattern in the galactose-specific lectin 24K from the egg jelly of the Chinook salmon Oncorhynchus tshawytscha was determined, and its previously reported amino acid sequence was confirmed by mass spectrometry. A combination of tryptic digestion, HPLC separation, and chemical, modifications was used to establish the location of seven disulfide bonds and one pair of free cysteines. After proteolysis, peptides containing one or two disulfide bonds were identified by reduction and mass spectral comparison. MALDI mass spectrometry was supported by chemical modification (iodoacetamidylation) and in silico digestion. The assignments of disulfide bonds were further confirmed by mass spectral fragmentation studies including in-source dissociation (ISD) and collision-induced dissociation (CID). Lectins of comparable biochemical functions can be found in amphibian eggs as well. Those eggs are covered with glycoproteinaceous extracellular matrix, which is known as the zonae pellucidae (ZP). The ZP consists of three major glycoproteins referred to as ZPA, ZPB, and ZPC, which contain homologous regions named "ZP domains". The ZP domain is also found in other secretory glycoproteins. Trans -membrane domains present at the C -terminus of ZP glycoproteins are removed at furin-processing sites. However, the details of this processing are unclear because of the lack of information about the precise C -termini of ZP glycoproteins. In this study, the N -termini and C -termini of the glycoprotein gp 37 (ZPB, from gp 37 precursor) and gp 41 (ZPC, from gp 43) from the clawed South African toad ( Xenopus laevis ) were determined by mass spectrometric analysis. Our results suggest that the N -terminus and C -terminus of gp 41 are generated by oviductin-mediated cleavage at GSR 55 and GSR 367 and the C -terminus of gp 37 is generated by furin-mediated cleavage at CNT 457 . These findings shed light on the biochemical processing of gp 43 to gp 41 and gp 37 precursor to gp 37.

Pages

187

ISBN

9780542897665

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