The fact that men and women biologically, morphologically and physiologically are different [25], brings about the magnitude and nature of this difference at molecular level. It is well known that, genomically and genetically both sexes posses the same chromosomes and genes except for the sex chromosomes (XY), suggesting that the main differences between both sexes would be proteins encoded by or their synthesis is influenced by proteins/peptides (PPs) encoded by genes on Y chromosome e.g. SRY gene [26]. In this study, proteomic analysis revealed marked differences in levels of 20 PPs, none of which is encoded by genes located in the sex chromosomes, unlike what was observed elsewhere [27]. Furthermore, the PPs up regulation was more evident in women, confirming a recent report showing significant sexual dimorphisms in protein abundance between twin pairs of opposite sex [27], although men have the unique Y-chromosome.
The observation that, all identified PPs are expressed by genes located in the autosomal chromosomes (Table 2), is supported by a previous study reported that the sex differences in part are due to differences in expression of genes not in sex chromosomes [28], what was termed as sex-biased gene expression. Furthermore, three PPs known to be regulators for gene expression were differentially expressed in this study, supporting the sex-biased expression independent of the sex-chromosomes. Two transcription factors, Homeobox protein Hox-D13 and Isoform 4 of Coiled-coil domain-containing protein 17 (CCDC17) were up-regulated in women while a translational factor, Eukaryotic translation initiation factor 4E (eIF4E) type 3 (Fragment), was up-regulated in men. The Hox-D13 is not unexpected to be upregulated in women since the Hox genes family products are known to be transcriptional factors involved in female reproductive system development and function [29]. However, there are no clues that the other two PPs, the CCDC17 and eIF4E, have any specific sex-related role.
Interestingly, only one out of the 20 identified PPs was directly related to sex, the sex hormone-binding globulin (SHBG), which was up-regulated in women. This protein is only rarely investigated in healthy subjects. One study showed that SHBG mRNA is strongly correlating with serum SHBG protein level with higher levels of the mRNA and protein in women than in men [30]. This report is supporting our proteomic analysis finding.
Of the 20 PPs which were markedly differentially expressed between sexes, 11 were up regulated in women and 9 in men, however, the difference in abundance were more marked in PPs up regulated in women (Fig. 1A&B). Using the same material, we previously detected more than 2 million PPs in human serum [6], the extremely low number of the identified PPs in this study compared to the total number of PPs, can be explained by the set criteria for marked differentially expressed PPs. The system was set to select only markedly differentially expressed PPs (MFC ≥1.5), what might had excluded thousands of PPs differentially expressed between the 2 sexes but with fold change less than 1.5. Worth noting in biological systems function and homoeostasis depend on fine balance of molecules i.e. not necessarily the quantity [31, 32]. However, using proteomic platform the identified PPs were selected based on quantity not function.
In this study, we didn’t aim to elaborate on each of the 20 PPs individually, as the informatics databases are provided for that purpose (see materials and methods). Our goal here to, interprets the functional classes of the identified PPs, in terms of physiological and clinical differences between the two sexes. Worth noting, the identified PPs were restricted to a very limited classes of proteins based on their functions (Table 2).
A noticeable and relevant observation regarding the identified PPs, was the up regulation of Apo-lipoproteins, Apo AII and Apo CII in women and that of Apo B100 in men. The former two were the predominant Apo proteins in chylomicron and VLDL, which are not involved in atherosclerosis and coronary heart diseases directly (CHD), while the Apo-B100, the major protein in LDL, is the carrier of pathological cholesterol [33]. It is well known that men are more susceptible to CHD compared to women in the same age before menopause [8]. The sex-biased differential expression of the lipid metabolism class of PPs in this study is consistent with the inherent protection of women against CHD during the reproductive age.
The sex-based immunological dimorphism was known long time ago [34], however, detailed analysis showing the abundance of the immunoglobulins (Ig) chains at transcriptome or proteome level were rarely investigated. In this study, the up regulation of Ig mu chain C region (IgM heavy chain) in women and of Ig gamma-1 &2 chain regions (IgG heavy chains) and complement factor H (the complement alternative pathway) in men, probably reflects the difference between the two sexes in response to antigens. In line with our findings are Saudi and Iranian studies, both showed higher levels of IgM in healthy women compared with men, and higher IgG in men compared with women in the Saudi but not in the Iranian study [35, 36]. Studies of sex difference in the immune responses following infection/vaccination showed that, women across all age groups were able to mount higher immune responses to infections and vaccines than men [14, 37]. In contrast, women are at much higher risk for development of autoimmune diseases e.g., SLE compared to men [38], however, it worth to know if there is a link between the IgM and the increased risk in women for the above-mentioned conditions.
To the best of our knowledge, the differences in levels of the acute phase proteins, alpha-1-cid glycoprotein -1 and 2 (AGP1 and 2), also known as orosomucoid, between men and women were not reported before. In this study both PPs were up-regulated in women. The associations of AGP with rheumatoid arthritis [39] and other autoimmune diseases e.g. Grave’s disease [40], together with the high prevalence of these disorders in women [11] are strongly supporting the upregulation of AGP in women observed in this study. The Multiple PDZ domain protein, was the only signal transduction PPs identified in this study, it was up-regulated in women. This protein is known to have the highest expression level in corpus callosum (brain). The up-regulation of the PDZ domain in women worth further investigations because, of the associations of this PP with several and diverse pathologies [41], and it is also involved in control of cell polarity and signal transduction [42, 43].
In this study, 9 out of the 20 PPs were upregulated in men compared with women, but the up regulation in men was not as high as in women in terms of abundance (fold change). Of the noticeable upregulated PPs in men, the 2 connective tissue (CT) proteins, fibronectin and vitronectin, the sex-differences in the levels of both PPs was not reported before. However, both proteins were noticed to be upregulated in T2DM [6, 44]. Furthermore, men and women differ in musculoskeletal system in which CT proteins are major component [45]. The differences in CT proteins between the two sexes have clinical implications e.g. the incidence of the anterior cruciate ligament injury is almost 10 times higher in women compared with men performing the same activity [46], an observation that supports our findings.
The last PP up-regulated in men is the Isoform 2 of Putative golgin subfamily A member 2B, one of the Golgi apparatus proteins, which is probably encoded by a pseudo-gene, (https://www. genecards.org/cgi-bin/carddisp.pl?gene=GOLGA2P5). However, no single article was published about this PP in the Pub Med database. This and other PPs listed in (Table 2), are not discussed here, but worth further investigation.
Finally, as a summary for the above, we further explored the functional characteristics and relatedness of the 20 PPs to disease and other immune mediated disorders using Ingenuity Pathway Analysis (IPA), (Table 3 & Fig. 4). The principle of the IPA was explained previously [47]. Only 14 of the 20 PPs were mapped in the IPA database and were found to be implicated in multiple signaling networks including cell-to-cell signaling and interaction, lipid metabolism, and small molecule biochemistry. The functional annotations of these proteins with others as transporter, transmembrane receptor, and catalysts is referred to in (Table 3), while other functions included implications in different drug agents (data not shown).
In conclusion, 20 PPs were found to be differentially expressed in healthy men and women, 11 up-regulated in women with high fold change, while 9 were up-regulated in men with lower fold change compared to women. Although differentiating between the two sexes, none of the identified PPs is encoded by gene allocated in sex chromosomes. The frequently obvious links between sex, PPs up-regulation, and disease susceptibility e.g. the Apo-B100, men and CHD, are validation for this proteomic data. The association of the remaining PPs with sex and their physiological and pathological roles need further investigations. Finally, the molecular basis of the phenotypic differences between men and women is still unclear.