This application proposes studies of bacterial IgA proteases, enzymes that specifically cleave secretory IgA, the major antibody in saliva and other secretions. Since IgA cleavage products may accelerate bacterial colonization, the enzymes of both commensal and pathogenic bacteria are immuno-evasive. Research emphasis will be on Streptococcus sanguis. When tested in vitro these early colonizers of the tooth surface have fully active IgA protease bound to the cell surface, producing IgA cleavage directly at the cell wall.
The research aims are: 1) Crystallization of IgA protease, intended to identify the basis for their remarkable substrate specificity by structural analysis of the enzyme conformation and active site. This will be done with Dr. Edward Baker, a highly experienced protein crystallographer; 2) Identification of the human IgA components needed for enzyme binding to develop substrate-based inhibitors, and substrates other than human IgA. These experiments will employ a novel expression cloning of the IgA alpha chain as a fusion protein with gastrointestinal peptide hormone receptors. In this form the substrate will be exposed for access by the large IgA protease enzymes, and will allow site specific and deletional mutagenesis; and 3) Examination of the mechanism of streptococcal IgA protease cell binding by using lactoferrin (LF), a ubiquitous, pre-immune protein which acts directly on bacterial cells to dissociate the active surface enzyme. This extraction is selective, and may be an important host defense mechanism during the earliest phases of plaque formation. Removal of the membrane proteins by salivary lactoferrin and its derivatives from the small numbers of S. sanguis cells that initiate plaque will be studied. Extraction will be measured and analyzed by epitope tagging of protease or protease precursors.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE009677-08
Application #
6137914
Study Section
Oral Biology and Medicine Subcommittee 1 (OBM)
Program Officer
Mangan, Dennis F
Project Start
1991-02-01
Project End
2002-12-31
Budget Start
2000-01-01
Budget End
2000-12-31
Support Year
8
Fiscal Year
2000
Total Cost
$276,893
Indirect Cost
Name
Tufts University
Department
Type
DUNS #
079532263
City
Boston
State
MA
Country
United States
Zip Code
02111
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