SPANX-A/D protein subfamily plays a key role in nuclear organisation, metabolism and flagellar motility of human spermatozoa

Abstract

Human sperm protein associated with the nucleus on the X chromosome (SpAnX) genes encode a protein family (SpAnX-A,-B,-c and-D), whose expression is limited to the testis and spermatozoa in normal tissues and to a wide variety of tumour cells. present only in hominids, SpAnX-A/D is exclusively expressed in post-meiotic spermatids and mature spermatozoa. However, the biological role of the protein family in human spermatozoa is largely unknown. combining proteomics and molecular approaches, the present work describes the presence of all isoforms of SpAnX-A/D in human spermatozoa and novel phosphorylation sites of this protein family. In addition, we identify 307 potential SpAnX-A/D interactors related to nuclear envelop, chromatin organisation, metabolism and cilia movement. Specifically, SPANX-A/D interacts with fumarate hydratase and colocalises with both fumarate hydratase and Tektin 1 proteins, involved in meeting energy demands for sperm motility, and with nuclear pore complex nucleoporins. We provide insights into the molecular features of sperm physiology describing for the first time a multifunctional role of SPANX-A/D protein family in nuclear envelope, sperm movement and metabolism, considered key functions for human spermatozoa. SpAnX-A/D family members, therefore, might be promising targets for sperm fertility management. Currently, over 186 million people worldwide have infertility problems 1 , and male factors represent approximately 40-50% of clinical infertility cases 2. Among other factors, anatomical or genetic abnormalities, problems in spermatogenesis or environmental factors 3 , are responsible for abnormal sperm parameters which contribute to male infertility. However, approximately 20-30% of men with normal sperm parameters have impaired fertility suggesting that male infertility can be caused by different deficiencies not yet described. The sperm protein associated with the nucleus mapped to the X chromosome (SPANX) family is a testis-specific multigene family. SPANX genes encode proteins that belong to the so-called "cancer testis antigens" (CTA) family, a group of proteins whose expression is limited to the testis and spermatozoa in normal tissues and to a wide variety of tumours 4,5. The SPANX family includes two subfamilies: the SPANX-N subfamily, composed of SPANX-N1,-N2,-N3,-N4 (in the locus Xq27.3) and-N5 (in the locus Xp11.22) that share a high level of sequence homology 6 ; and the SPANX-A/D subfamily, which includes SPANX-A1,-A2,-C,-D (in the locus Xq27.2) and-B (in the locus Xq27.1) with high sequence homology 5,7. Evolved from a common ancestor, the SPANX-N gene family is present in mice, rats and all primates, while the SPANX-A/D family is expressed only in hominids, including humans, bonobos and chimpanzees 8. All SPANX proteins exhibit a similar post-meiotic expression pattern during spermatogenesis, appearing for the first time in haploid spermatids 4,7,9. In mature spermatozoa, SPANX-N proteins are localised on the acrosome 6 , while SPANX-A/D proteins are expressed as dots

The multifunctional role of SPANX-A/D protein subfamily in the promotion of pro-tumoural processes in human melanoma

“…However, surprisingly, we could not find any phosphorylated SPANX-A/D residues at the NLS in A375 cells. Considering that the SPANX-A/D protein family is mainly localised in the nucleus of melanoma cell lines, our findings confirm that the phosphorylation state of the NLS is not crucial for nuclear SPANX-A/D translocation either in spermatozoa 7 or in A375 cell lines. SPANX-A/D proteins play a role in the invasion and/or metastasis of several tumours.…”

“…Hence, proteomic analysis corroborated the presence of SPANX-A/D in A375 cells but could not determine whether one, two, three or all members were co-expressed. According to our results, SPANX-A/D can be phosphorylated in human spermatozoa 7 . To uncover whether this subfamily is also phosphorylated in A375, we conducted proteomic studies.…”

“…However, the SPANX-A/D protein family may be involved in other uncharacterised biological functions in melanoma cells. According to our proteomic analysis, the SPANX-A/D protein family plays a multifunctional role in melanoma cells, as we have previously reported in human spermatozoa 7 . Considering that this protein family is mainly expressed in the nucleus of the A375 melanoma cell line, SPANX-A interactors that are involved in nuclear functions show the highest intensity values.…”

“…Although higher levels of SPANX-A/D are reported to be associated with more aggressive skin tumours 9 , our results show the presence of the SPANX-A/D protein family in both primary and metastatic melanoma cell lines presenting nuclear localisation. However, we have previously reported that the NLS of SPANX-A/D proteins is strongly phosphorylated in human spermatozoa 7 . However, surprisingly, we could not find any phosphorylated SPANX-A/D residues at the NLS in A375 cells.…”

“…Plasmids and transfection. The plasmid encoding YFP-SPANX-A has been previously described 7 . For transfection experiments, A375, MelHO and Colo-800 melanoma cells were seeded onto 12-well or six-well tissue culture plates.…”