PMC:3431878 / 6380-12315
Annnotations
{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/3431878","sourcedb":"PMC","sourceid":"3431878","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3431878","text":"3. Nucleotide and Amino Acid Sequence\nThe currently available genomic information on CA125 is based on two autonomous studies conducted in 2001 [17,18]. By screening a cDNA library generated using OVCAR-3 mRNA, Yin et al. identified a clone with a 5797 base pair (bp) nucleotide sequence [17] (reviewed in [19,20]). Although this region translated to a partial CA125 protein, it confirmed and revealed the existence of some key features. Importantly, they established the presence of partially conserved tandem repeats and were the first to describe the homology to the “sea urchin sperm protein, enterokinase, agrin” (SEA) domain. Yin et al. also postulated the existence of a transmembrane domain based upon a cluster of hydrophobic residues and a phosphorylation site based on a tyrosine phosphorylation consensus motif [17]. Furthermore, O-glycosylation was inferred due to the serine, threonine and proline content as well as N-glycosylation due to the asparagine content of the protein. These findings were supported and extended on by O’Brien and co-workers [18,21]. Here, a large transcript encoding CA125 was sequenced based upon a cyanogen bromide cleavage fragment of CA125 and a translated expressed sequence tag [16]. The resultant protein was described in three parts; an amino terminal domain, a repeat domain and a carboxy terminal domain (Table 1, Figure 1). The carboxy terminal domain reported by O’Brien et al. was homologous to the findings of Yin et al. [17,18,20]. One feature described only by O’Brien was the existence of a potential proteolytic cleavage site at amino acid residues 171–181 [18].\nAs described previously, the repeat domain consists of partially conserved tandem repeats that are 156 amino acids in length. The SEA domain was noted to constitute the first 131 amino acids of this region [18]. However, the amino acid sequence exhibiting a SEA domain is variable in length as the main feature of the consensus sequence is a “strand-strand-helix-strand-strand-helix” secondary structure motif and is unaffected by small sequence gaps between these units [24]. Therefore, as most CA125 tandem repeats are polymorphic, the SEA domain may not necessarily be 131 amino acids long. This is exemplified by the identification of a SEA domain consensus sequence with a length of 59 amino acids located in the non-tandem repeat region of the C-terminal domain [17]. Furthermore, the repeat domain contains a highly conserved methionine at position 24 as well as two highly conserved cysteine residues at position 59 and 79 [18]. These cysteine residues in the repeat domain were hypothesized by O’Brien to form a 19 amino acid loop to which the M11 antibody binds. Additionally, O’Brien demonstrated a loss of binding of the M11 antibody to a recombinant repeat domain when digested with Asp-N or Lys-C as this would have disrupted the cysteine loop [18].\nIn the study by O’Brien et al., 45 unique and 15 redundant tandem repeat sequences were identified [18]. It was concluded that CA125 consists of at least 60 tandem repeats. However, due to difficulties associated with sequencing repeat regions some may be wrongly positioned or unknown, leaving the exact number of repeats undefined [18]. Furthermore, O’Brien states that the genome database is deficient in repeat information as repetitive sequences were potentially deleted during the compilation process of the human genome [18]. This suggests that the currently available nucleotide sequence of CA125 may not be correct.\nThe amino terminus was first described as a 1637 amino acid domain coded by 5 exons [17,18]. This was later revised by O’Brien et al. where an additional 37,700 nucleotides were sequenced from chromosome 19 [21]. Acquisition of the complete amino domain was confirmed by the identification of a Kozak translational initiation sequence as well as supporting Northern blot and mRNA expression data [21]. The main feature of this domain is the abundance of serine and threonine residues, which represent many potential sites of O-glycosylation, and disperse asparagine residues as potential sites of N-glycosylation [21].\nThe elucidation of the almost complete CA125 nucleotide sequence represented a significant leap in understanding. It enabled clarification of known and/or suspected features such as glycosylation (amino domain), multivalent antibody binding (repeat domain) as well as anchorage to and release from the membrane (carboxy domain) [12,27]. The findings also raise questions regarding the generation of lower molecular weight isoforms. Only the existence of a CA125 precursor protein of approximately 400 kDa has been found to exist in the cytoplasm of OVCAR-3 cells prior to protein maturation [28]. CA125 variants are also thought to arise from proteolytic cleavage. Proteins such as MUC1 have a proteolytic cleavage motif, GSVVV, in their SEA domain [29,30], however the presence of a motif with a similar consensus sequence has only been confirmed in one SEA domain of CA125 thus far [22]. A cluster analysis of a human CA125 sequence, containing the first 12 tandem repeats and a murine homolog to the C-terminus of CA125, was performed by Maeda et al. [22]. Interestingly, a DSVLV sequence in the second SEA domain from the cytosolic side of CA125 was clustered with the GSVVV sequence. Therefore, it was concluded that proteolytic cleavage might also occur in this domain of CA125 [22]. However, the formation of further proteolytic fragments of CA125, by the mechanism of the DSVLV or GSVVV sequence, in additional SEA domains has not been reported. Furthermore, O’Brien et al. suggested that the formation of alternative splicing products of this protein seems unlikely, based on the inherent variation of the five exons encoding for each repeat sequence [18]. Therefore, a discrepancy exists between antibody findings of low molecular weight isoforms and the protein sequence data (discussed 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