All antibodies (Abs) are glycoproteins and all heavy chains have at least one N-linked carbohydrate in their constant regions. Many Abs also have variable (V) region associated carbohydrate. The proposed studies will systematically investigate the role and function of the Ab associated carbohydrate by altering its presence, position and structure. Central to this investigation is the ability to produce large quantities of antibodies possessing altered carbohydrates of defined structure using the well- characterized glycosylation mutants of Chinese hamster ovary (CHO) cells. A significant percentage of Abs contain variable region carbohydrate and although V region carbohydrate can influence Ag binding, the function of V region carbohydrate remains incompletely understood. A family of Abs of the same specificity with carbohydrate at different positions in their V region will be produced and their functional properties including in vitro half-life, reactivity with rheumatoid factors (RFs) and affinity for antigen (Ag) determined. Site-directed mutations will be used to ascertain which V region sequences can be glycosylated. The presence of carbohydrate in Ch2 is essential for the biological activity of IgG. The carbohydrate is always biantennary with heterogeneity in structure normally observed. Evidence suggests that certain glycosylation patterns can contribute to aberrant immunoglobulin activity with a high incidence of agalactosylated Abs observed in rheumatoid arthritis (RA). Abs with altered carbohydrate structure will be produced to determine if changes in carbohydrate structure result in changes in Ab function including half-life, the ability to bind cellular receptors for Fc and to activate complement by either the classical pathway or through mannose binding proteins. Recognition by lectins, monoclonal antibodies, and Proteins A and G, will be used to probe alterations in peptide conformation and carbohydrate accessibility. In addition to giving insights into Ab function, these experiments will elucidate the role of glycosylation alterations in creating pathogenic Abs. IgA is the important isotype in providing specific protection at the mucosal surface. The role of carbohydrate in IgA function will be investigated by altering both the presence and structure of its associated N-linked carbohydrate. The contribution of the O-linked carbohydrate of IgA1 will also be assessed and the role of the N-linked carbohydrate of J- chain investigated. The recombinant molecules will be evaluated for their in vivo half-life and biodistribution as well as their resistance to bacterial proteases, binding to bacterial and cellular receptors, ability to activate the alternative complement pathway and to bind to the polymeric Ig receptor and be transported across the epithelium. The information obtained may permit the design and production of IgA more effective in passive protection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AI029470-07
Application #
2390337
Study Section
Allergy and Immunology Study Section (ALY)
Project Start
1990-12-01
Project End
1999-03-31
Budget Start
1997-04-01
Budget End
1998-03-31
Support Year
7
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Kim, Yun Cheol (2011) Introducing predetermined mutations throughout a target gene using TDEM (transposon-directed base-exchange mutagenesis). Methods Mol Biol 705:275-93
Yoo, Esther M; Yu, Li J; Wims, Letitia A et al. (2010) Differences in N-glycan structures found on recombinant IgA1 and IgA2 produced in murine myeloma and CHO cell lines. MAbs 2:320-34
Kim, Yun Cheol; Lee, Hui Sun; Yoon, Sukjoon et al. (2009) Transposon-directed base-exchange mutagenesis (TDEM): a novel method for multiple-nucleotide substitutions within a target gene. Biotechniques 46:534-42
Chintalacharuvu, Koteswara R; Gurbaxani, Brian; Morrison, Sherie L (2007) Incomplete assembly of IgA2m(2) in Chinese hamster ovary cells. Mol Immunol 44:3445-52
Beenhouwer, David O; Yoo, Esther M; Lai, Chun-Wei et al. (2007) Human immunoglobulin G2 (IgG2) and IgG4, but not IgG1 or IgG3, protect mice against Cryptococcus neoformans infection. Infect Immun 75:1424-35
Gurbaxani, Brian Mohan; Morrison, Sherie L (2006) Development of new models for the analysis of Fc-FcRn interactions. Mol Immunol 43:1379-89
Dela Cruz, Jay Soriano; Trinh, Kamh Ryan; Chen, Hsiao Wen et al. (2006) Anti-HER2/neu IgG3-(IL-2) and anti-HER2/neu IgG3-(GM-CSF) promote HER2/neu processing and presentation by dendritic cells: implications in immunotherapy and vaccination strategies. Mol Immunol 43:667-76
Helguera, Gustavo; Dela Cruz, Jay S; Lowe, Christine et al. (2006) Vaccination with novel combinations of anti-HER2/neu cytokines fusion proteins and soluble protein antigen elicits a protective immune response against HER2/neu expressing tumors. Vaccine 24:304-16
Gurbaxani, Brian; Dela Cruz, Linh L; Chintalacharuvu, Koteswara et al. (2006) Analysis of a family of antibodies with different half-lives in mice fails to find a correlation between affinity for FcRn and serum half-life. Mol Immunol 43:1462-73
Dela Cruz, Jay S; Morrison, Sherie L; Penichet, Manuel L (2005) Insights into the mechanism of anti-tumor immunity in mice vaccinated with the human HER2/neu extracellular domain plus anti-HER2/neu IgG3-(IL-2) or anti-HER2/neu IgG3-(GM-CSF) fusion protein. Vaccine 23:4793-803

Showing the most recent 10 out of 70 publications