Solution Structure of the SEA Domain from the Murine Homologue of Ovarian Cancer Antigen CA125 (MUC16)

Abstract

Human CA125, encoded by the MUC16 gene, is an ovarian cancer antigen widely used for a serum assay. Its extracellular region consists of tandem repeats of SEA domains. In this study we determined the three-dimensional structure of the SEA domain from the murine MUC16 homologue using multidimensional NMR spectroscopy. The domain forms a unique ␣/␤ sandwich fold composed of two ␣ helices and four antiparallel ␤ strands and has a characteristic turn named the TY-turn between ␣1 and ␣2. The internal mobility of the main chain is low throughout the domain. The residues that form the hydrophobic core and the TY-turn are fully conserved in all SEA domain sequences, indicating that the fold is common in the family. Interestingly, no other residues are conserved throughout the family. Thus, the sequence alignment of the SEA domain family was refined on the basis of the three-dimensional structure, which allowed us to classify the SEA domains into several subfamilies. The residues on the surface differ between these subfamilies, suggesting that each subfamily has a different function. In the MUC16 SEA domains, the conserved surface residues, Asn-10, Thr-12, Arg-63, Asp-75, Asp-112, Ser-115, and Phe-117, are clustered on the ␤ sheet surface, which may be functionally important. The putative epitope (residues 58 -77) for anti-MUC16 antibodies is located around the ␤2 and ␤3 strands. On the other hand the tissue tumor marker MUC1 has a SEA domain belonging to another subfamily, and its GSVVV motif for proteolytic cleavage is located in the short loop connecting ␤2 and ␤3.CA125 is a serum marker that is widely used to monitor ovarian cancer because it is overexpressed in ovarian cancer cells and secreted into the blood. An elevated serum CA125 level is a useful indicator of ovarian cancer, but it is also observed in a number of benign conditions (1, 2). CA125 is a mucin-type O-linked glycoprotein (3, 4), but other details about its molecular nature remain unclear. Recently two research groups cloned CA125 (5-8), revealing that CA125 is a membrane protein with some splicing variants. The splicing variants have the same intracellular and transmembrane regions. The extracellular domain consists of the SEA 1 domains, which are repeated 7, 12, or 60 times, according to the variant. The gene was named MUC16, after the mucin-like nature of CA125. The elucidation of the amino acid sequence has made it possible to specify the approximate position of the epitope. A previous study showed that the peptide epitope position of CA125 is located between two conserved cysteines in the SEA domain (7).A cDNA of the murine MUC16 homologue, cloned in the RIKEN FANTOM project (9), has a total of 258 amino acids and a transmembrane domain. It is 66% identical to the C terminus of human MUC16 and has only one SEA domain in its extracellular region. However, our investigation of the mouse and human genomic sequences showed that they share the same characteristic repeat structure of MUC16. Thus, the murine MUC16 appears to have splicing variants, as i...

Effect of Immune Complex Formation on the Distribution of a Novel Antibody to the Ovarian Tumor Antigen CA125

“…In some cases, the antigen is shed from the cell surface into the circulation where the antibody may form ICs with the circulating antigen, potentially affecting therapeutic efficacy by preventing it from reaching the tumor and/or altering the antibody kinetics of distribution and clearance (Bruno et al, 2005). A number of ovarian cancer preclinical and clinical studies have addressed the potential risks of shed antigen (Hagan et al, 1985;Haisma et al, 1987;Pimm et al, 1989;Kobayashi et al, 1993;Pimm, 1995;Sakahara et al, 1996;Davies et al, 1997;McQuarrie et al, 1997;Prinssen et al, 1998;Maeda et al, 2004). In patients with ovarian cancer, IC formation was detected upon delivery of an anti-CA125 antibody.…”

Analysis of CA125 antigen in normal human seminal plasma highlightsthe molecular heterogeneity of underlying glycosylated species

“…MUC16 can be cleaved between the N-and C-terminal fragments, possibly in the SEA (sea urchin sperm protein, enterokinase, and agrin) domain, with potential influence on immunoreactivity, since both epitopes for OC125 and M11-like antibodies contain parts of two consecutive SEA domains with a linker (Maeda et al, 2004;Blalock et al, 2008). So far, proteomic profiling identified a 2351.2 kDa protein/spot as high molecular mass MUC16/CA125 (Milardi et al, 2012), but a CA125-immunoreactive band at 130 kDa was also observed in another immunoblot study (Halila, 1985).…”

Conflicting Views on the Molecular Structure of the Cancer Antigen CA125/MUC16

“…Amino acid analysis of MUC16 revealed that its sequence contains numerous lysine and arginine residues that are remote from the postulated O-glycosylation sites [58]. The crystal structure of the repeats was characterized, revealing the presence of varying types of SEA (S = sea urchin, E = enterokinase, and A = agrin) domains, a structural motif found in highly O-glycosylated membrane proteins that is cleaved and then reassociates via noncovalent bonds [62].…”

“…Of additional note, MUC16 codes splicing variants that give rise to polypeptides that differ in the number of SEA domains. Consequently, CA125 antigen may have 7, 20, or 60 tandem repeats (TR) increasing the core molecular weight from 200000 daltons to almost 4 million daltons [62].…”

“…an additional 10431 amino acids have been found. The tandem repeat domain consists of 40-60 repeats, each 156 amino acids long with a disulphide bridge loop of 19 amino acids [42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57]59,[59][60][61][62][63]. Although the overall structure is well conserved, only few repeats have identical amino acid sequences.…”