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The perplexity of calcium-binding protein, spermatid-associated 1 (CABS1): A molecule that despite its name, is present beyond the reproductive tract, with ties to stress, and possessing an anti-inflammatory domain only preserved in simians
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- Author / Creator
- Reyes Serratos, Eduardo
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A protein in rat submandibular glands (SMG) led our group to research human calcium-binding protein, spermatid associated 1 (CABS1). In a model of lung inflammation in rats where neural input was interrupted, our group observed that SMG released an anti-inflammatory factor later identified as Submandibular rat protein 1 (SMR1). Rat SMR1 was present in serum, saliva, testes, and SMG. A seven amino acid peptide near the carboxyl terminus of SMR1, TDIFEGG, was responsible for the anti-inflammatory activity. Synthetic TDIFEGG and derivates had anti-inflammatory activity in several animal models, but a human trial in mild atopic asthma showed no significant effect. SMR1 is absent in humans, but we postulated that an analogous pathway existed. Thus, our group looked for a human protein that contained a similar peptide sequence, ultimately finding CABS1, which has the seven-peptide sequence, TDIFELL, also near the carboxyl terminus.
CABS1 has been characterized previously in testes of rat, mouse, and pig. Transcript has been found in testes, uterus, and salivary glands of mammals. Previously, our group showed CABS1 protein in human (h) SMG, lungs, testes, and saliva via Western blots (WB) using a single polyclonal antibody. The data suggested that hCABS1 exists as several variants of different molecular weight. A 27 kDa band in saliva positively correlated to perceived psychosocial stress, and bands <27 kDa could be indicators of resilience to stress. Based on these data, my hypotheses were that hCABS1 is present beyond the testes, in SMG, saliva and blood, like rat SMR1, and that variants of CABS1 in saliva are biomarkers of stress.
To extend our previous findings, we analyzed WB profiles using four pAbs to hCABS1 and overexpression cell lysate controls, SMG, and saliva. We used these profiles as guidance to isolate in-gel sections that we speculated contained hCABS1 and evaluated their protein content through mass spectrometry sequencing (MS-seq). hCABS1 was detected in overexpression cell lysates via MS-seq, yet contrary to what our WB profiles, to date we have not detected the protein in human-derived biofluids or tissue extracts.
Given the limiting amounts of pAbs available, a capillary nano-immunoassay (CNIA) was used to validate the occurrence of hCABS1 variants in saliva. With this platform, we successfully validated our previous stress-related findings, and also reported another variant of hCABS1 (60 kDa) in saliva detected by a pAb that targets TDIFELL. CNIA optimization also led to improvements in our WB protocol that encouraged reevaluation of our previous data in saliva. WB analysis using pAbs allowed us to target sites in one and two-dimensional gels for MS-seq analysis. However, efforts to confirm the presence of hCABS1 in saliva using MS-seq were unsuccessful, perhaps because of limitations in our pAbs or the amount of hCABS1. We speculated that the pAb detected hCABS1 and potentially other protein(s), and thus developed monoclonal antibodies (mAbs) targeting three domains of the protein. Using these mAbs, WB analyses of overexpression cell lysate controls, SMG, saliva and serum detected fewer bands than pAbs, but validated that hCABS1 is present beyond the testicular compartment.
We also conducted immunohistochemical (IHC) analyses of SMG with four pAbs and one mAb, showing that hCABS1 is present in the cytoplasm of duct cells of SMG. IHC of human testis, suggest that hCABS1 is present in cytoplasm of primary spermatogonia in seminiferous tubules and outside the tubules in Leydig cells. Moreover, we have begun to immunoprecipitate hCABS1 from a transient overexpression cell lysate with our mAbs, and have confirmed success using MS-seq.
We have further dissected the protein using in silico analyses. These studies provide evidence of disordered domains close to the carboxyl terminus of hCABS1, where its anti-inflammatory domain is located, as well as in other segments of the protein. A phylogenetic analysis showed that the peptide sequence, TDIFELL, is found exclusively in primates of the infraorder Simiiformes, which includes humans. Supporting our WB work, a predicted degradome analysis of hCABS1 with neutrophil elastase identified several potential cleavage sites, theoretically generating hCABS1-derived peptides. Altogether, evidence in this dissertation supports the presence of hCABS1 beyond the testes, in SMG and blood. However, evidence about hCABS1 in saliva is inconclusive and thus the link to stress remains to be clarified. -
- Graduation date
- Fall 2022
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- Type of Item
- Thesis
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- Degree
- Doctor of Philosophy
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- License
- This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.