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Dissertation
Primate [alpha]1,3-galactosyltransferase: structure-function analysis and application for the enhancement of vaccine immunogenicity
Doctor of Philosophy (Ph.D.), Medical College of Pennsylvania
Sep 1996
DOI:
https://doi.org/10.17918/00007247
Abstract
The glycosyltransferase enzyme [alpha]1,3-galactosyltransferase ([alpha]13GT) is active in nonprimate mammals and New World monkeys, but absent in Old World monkeys, apes, and humans. The latter species produce large amounts of natural antibody, termed anti-Gal, which interact specifically with [alpha]-galactosyl epitopes, the biosynthetic product of [alpha]13GT. Our objectives were to study structure-function relationships in this enzyme, and based on the information obtained, to express large amounts of recombinant [alpha]13GT. We planned to use this recombinant enzyme for modulation of the carbohydrate makeup of influenza virus vaccines to enhance their immunogenicity. Our basic hypothesis is that vaccine antigens which display de novo synthesized [alpha]-galactosyl epitopes can form in situ immune complexes with anti-Gal, and be effectively targeted for uptake, processing, and presentation by FcR-bearing antigen presenting cells. We have cloned the cDNA for [alpha]13GT from the marmoset, where the enzyme is active, and conducted a truncation analysis of the coding region. We show that up to 67 amino acids can be deleted from the N-terminal "stem region" of [alpha]13GT without affect on enzymatic activity. In contrast, the loss of as few as 3 amino acids from the carboxy terminus completely abrogated [alpha]13GT catalytic activity. Likewise, insertion of a coding frameshift common to all ape species, including humans, resulted in total loss of enzyme function. Sequence analysis of RACE and cDNA library PCR clones revealed that at least four isoforms of [alpha]13GT are expressed in the B958 marmoset cell line by alternative splicing of two noncoding exons within the 5' untranslated region of the enzyme, generating leader sequences which are heterogeneous in length. Based on our truncation analysis, we have been able to express a soluble recombinant form of the enzyme in the baculovirus system (r[alpha]13GT), which lacks the membrane anchoring domain. We show that r[alpha]13GT has the same acceptor specificity and similar kinetic properties compared to natural [alpha]13GT, and can readily synthesize [alpha]-galactosyl epitopes on influenza hemagglutinin which are bound by anti-Gal antibodies. In future studies we plan to use the method we have developed here to demonstrate the increased efficiency of vaccines which express de novo synthesized [alpha]-galactosyl epitopes.
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Details
- Title
- Primate [alpha]1,3-galactosyltransferase
- Creators
- Timothy R. Henion
- Contributors
- Uri Galili (Advisor) - Drexel University, Medical College of Pennsylvania (1970-1993)
- Awarding Institution
- Medical College of Pennsylvania
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Medical College of Pennsylvania; Philadelphia, Pennsylvania
- Number of pages
- xii, 194 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Medical College of Pennsylvania (1970-1993)
- Other Identifier
- 991021888862504721