3.3 The negative effect of this missense variant onCFAP47 expression and function
To investigate the impact of this variant on protein structure, PyMOL Viewer software was used to visualize the effects of altered residues on protein-structure models (Figure 4a). A predicted structure of CFAP47 with the residues 1-754. Mutant V472M showed it may affect the stability of the original β-sheet region for methionine occupied more space than valine. This may indicate the structure of CFAP47 was disordered. To further determine the impact of c.1414G>A on CFAP47expression, we detected CFAP47 expression in the spermatozoa of the two patients. CFAP47 expression was distributed in flagella in control spermatozoa, while CFAP47 staining was barely detected in the spermatozoa of patients (Figure 4c). Meanwhile, the western blotting showed similar results of significantly decreased protein expression of CFAP47 in the patients’ sperm lysates compared to the fertile control (Figure 4b).
As CFAP47 is regarded as an MMAF-associated gene (Liu et al., 2021), STRING analysis was used to predict which known MMAF genes CFAP47 may interact with. The result revealed that CFAP47 may be connected with CFAP69 (https://cn.string-db.org/) (Figure 5a). To confirm this assumption, co-immunoprecipitation was performed, and the result verified binding between CFAP47 and CFAP69 in human testis lysates (Figure 5b). In addition, a previous study demonstrated that CFAP47 regulated and interacted with CFAP65 (Liu et al., 2021). The expression levels of these two proteins were validated to be sharply reduced in the sperm of the two patients compared to the normal control via immunofluorescence staining and western blotting (Figure 5c). In addition, the immunofluorescence assay confirmed the colocalization of CFAP47 with CFAP65 and CFAP69 in mouse (Figure S1) spermatogenic cells at different stages, as well as mouse testis sections (Figure S2). Collectively, the interactions between CFAP47 and its interactors may play a pivotal role in sperm morphology.