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Published on: March 24, 2017
Peptide Mapping and Glycoanalysis of Cancer Cell-Expressed Glycoproteins CA215 Recognized by RP215 Monoclonal
1Andrology Laboratory, UBC Center for Reproductive Health, Vancouver, Canada.
Abstract:
RP215 monoclonal antibody was shown to react with carbohydrate-associated epitope(s) in cancer cell-expressed glycoproteins known as CA215 based on indirect experimental evidences. Efforts have been made to identify glycans in CA215 that may be involved in the epitope recognition. More than 100 tryptic peptides, derived from affinity-purified CA215, consist mainly of immunoglobulin superfamily (IgSF) proteins (~60%), mucins (~7%), and others. Glycoanalysis was performed with affinity-purified CA215 from two cancer cell lines, including (1) N- and O-linked glycan profilings and linked glycoanalysis, (2) glycosylation site mappings, and (3) treatments with selected glycolytic enzymes. High mannose and complex bisecting structures with terminal sialic acid (NeuAc or NeuGc) were detected in N-glycans, whereas as many as 10 O-glycans structurally similar to those of mucins were identified. Through glycosylation site mappings, two N-linked and six out of eight O-linked glycans were detected and matched almost 100% with human immunoglobulin heavy chains. Treatments with several glycolytic enzymes were found to have little effect on the immunoactivity of the RP215-epitope. The same activity was also not affected by the cancer cell culture in human serum instead of bovine serum, indicating that NeuAc and NeuGc are not involved in epitope recognition. The immunoassay results also suggested that the affinity-purified cancer cell-expressed immunoglobulins revealed similar structures and immunoactivities to those of normal human immunoglobulins, except that two additional O-glycans were detected in the former. Supplemental materials are available for this article. Go to the publisher's online edition of Journal of Carbohydrate Chemistry to view the free supplemental file.
Insights
The RP215 antibody recognizes cancer-associated antigen CA215, primarily on immunoglobulin superfamily proteins. Glycan analysis identified specific N- and O-linked structures, but these did not appear essential for antibody binding.
Area of Science:
- Biochemistry
- Immunology
- Glycobiology
Background:
- The RP215 monoclonal antibody targets carbohydrate epitopes on cancer-associated antigen CA215.
- CA215 is expressed on cancer cells and its exact glycan structure recognized by RP215 is not fully elucidated.
Purpose of the Study:
- To identify the specific glycans on CA215 recognized by the RP215 antibody.
- To characterize the glycosylation patterns of CA215 from cancer cell lines.
Main Methods:
- Affinity purification of CA215 from cancer cell lines.
- Glycoanalysis including N- and O-linked glycan profiling, glycosylation site mapping, and enzymatic treatments.
- Immunoassay to assess the effect of glycan modifications on antibody binding.
Main Results:
- CA215 consists mainly of immunoglobulin superfamily proteins and mucins.
- Identified diverse N-glycans (high mannose, complex bisecting) and 10 O-glycans.
- Two N-linked and six O-linked glycans matched human immunoglobulin heavy chains.
- Enzymatic treatments and culture conditions did not affect RP215 immunoactivity, suggesting terminal sialic acids are not critical for epitope recognition.
Conclusions:
- The RP215 epitope on CA215 is associated with specific N- and O-linked glycans, particularly those found on human immunoglobulin heavy chains.
- Terminal sialic acids (NeuAc, NeuGc) are unlikely to be the primary components of the RP215 epitope.
- Cancer cell-expressed immunoglobulins share glycosylation similarities with normal immunoglobulins, with minor differences observed.
