Monoclonal antibodies specific to the putative cancer signaling protein, Uev1A
Date
2008
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
Type
Degree Level
Masters
Abstract
UEV1A and MMS2 are two human genes whose proteins share greater than ninety percent sequence identity. Both Uev1A and Mms2 are ubiquitin-conjugating enzyme variants (Uev) that lack the active cystine residue required for the ubiquitin conjugation reaction. They work with the ubiquitin-conjugating enzyme Ubc13 to create Lys63-linked polyubiquitin chains which have recently been found to cause cellular signals not involving target protein degradation. Only the Mms2-Ubc13 complex functions in DNA repair in mammalian cells. In contrast, only the Uev1A-Ubc13 complex is involved in TRAF2- and TRAF6-mediated NF-¦ÊB activation by ubiquitinating NEMO/IKKg. UEV1B is a splice variant of UEV1A containing different N-terminal coding sequences and its cellular function is currently unknown. The NF-¦ÊB signaling pathway has been regarded as a primary pro-survival and anti-apoptotic response. UEV1A expression is positively correlated to tumor formation, suggesting that it plays a role in tumorigenesis. Furthermore, experimental overexpression of UEV1A alone is sufficient to activate NF-¦ÊB, which is reversible upon suppression of UEV1A expression by RNA interference. Overexpression of UEV1A also protects cells from stress-induced apoptosis and confers a growth advantage under serum-deprived conditions. These observations collectively support UEV1A as a candidate proto-oncogene. The objective of this research was to obtain monoclonal antibodies (Mabs) capable of recognizing Uev1, but not Mms2. This is a challenging task given very few possible epitopes that may distinguish the two proteins. The four sequences unique to Uev1A are located in the 30 a.a. N terminal region, the single substitution at a.a. 104, the 14 a.a. core region from a.a. 116-129 (7/14 variable) and the C-terminus at a.a. 167-169.Hybridoma cells were produced vis ¨¢ vis fusions of B cells from Uev1A-immunized mice with FO myeloma cells lacking the ability to produce immunoglobulin. The hybridoma cells were screened using enzyme immunoassays (EIAs) for reactivity with Uev1A and Mms2. Ascities fluid was produced for five Mabs named LN1, LN2, LN2A, LN2B and LN3. EIA and Western blotting of Uev1A-deletion constructs revealed that Mab LN1 binds specifically to amino acids (a.a.) 10-30 of the unique Uev1A N-terminal sequence, that Mabs LN2, LN2A and LN2B bind specifically to the unique a.a. 110-130 region of Uev1A, and that Mab LN3 binds specifically to a.a. 30-44 of Uev1A common to Mms2. Competition analysis of unconjugated Mabs versus horse radish peroxidase (HRP)-conjugated Mabs for binding to Uev1A permitted epitope mapping for the five Mabs. The results indicate that Mabs LN1 and LN3 inhibit each other from binding to their distinct sequences which are spatially adjacent. Mabs LN2 and LN2B inhibit each other, but not Mab LN2A. Additionally, Mab LN2A is unable to inhibit Mabs LN2 or LN2B. In summary, Mabs have been aquired to three catergories which were originally desired: one to the unique N-terminus (Mab LN1), three to the unique core sequence (Mabs LN2, LN2B and LN2A), and one to the same region Ubc13 binds to Uev1A and Mms2 (Mab LN3). The potential applications of these Mabs are discussed.
Description
Keywords
Cancer, Antibody, Ubiquitination, Uev1A
Citation
Degree
Master of Science (M.Sc.)
Department
Microbiology and Immunology
Program
Microbiology and Immunology