Takasaki Nobuyoshi, Koya Yoshihiro, Yamashita Mamoru, Nawa Akihiro
Department of Obstetrics and Gynecology Collaborative Research, Bell Research Center, Nagoya University Graduate School of Medicine, Nagoya, Japan.
Bell Research Center for Reproductive Health and Cancer, Kishokai Medical Corporation, Aichi, Japan.
Cell Death Discov. 2024 Dec 18;10(1):499. doi: 10.1038/s41420-024-02252-4.
Polypeptide N-acetylgalactosaminyltransferase-like protein 5 (GALNTL5) was identified as a pp-GalNAc-T family gene. Nevertheless, GALNTL5 has no glycosyltransferase activity. In mice, Galntl5 expression is restricted to differentiating spermatids, and haploinsufficiency leads to immotile spermatozoa with an aberrant protein composition. Moreover, heterozygotic deletions of human GALNTL5 have been detected in patients diagnosed with asthenozoospermia (low sperm motility). Although these findings indicate that GALNTL5 is a functional molecule essential for mature sperm formation in mammals, the exact function of GALNTL5 in spermiogenesis remains unknown. To clarify this role, we established the mouse spermatocyte cell line GC-2spd(ts), which exhibits drug-inducible GALNTL5 expression. Interestingly, continuous GALNTL5 expression in the resultant cell lines caused apoptosis with cell shrinkage, and GALNTL5 was localized in the endoplasmic reticulum (ER) and was associated with two ER-resident chaperone proteins, calnexin and BiP (GRP78). Calnexin recognized and strongly bound to the N-glycans on GALNTL5 molecules modified in the ER. In contrast, ER-resident BiP likely attached to GALNL5 regardless of its glycosylation. GALNTL5 expression abolished the binding between calnexin and misfolded substrate proteins, indicating that GALNTL5 directly blocks calnexin function. Furthermore, the interaction between GALNTL5 and calnexin decreased the level of BiP protein, and consequently also the expression levels of proteins that are resident in the ER, Golgi apparatus, and cytoplasm. These reduced protein levels were confirmed by loss of calnexin or BiP function in the GC-2spd(ts) cell line using siRNA knockdown. Further, sustained expression of GALNTL5 resulted in cell structure changes, including the position of the cis-Golgi apparatus and alterations in the ER network. These results strongly suggest that GALNTL5 contributes to alteration of the cell structure specific to differentiating spermatids by blocking ER function.
多肽N - 乙酰半乳糖胺基转移酶样蛋白5（GALNTL5）被鉴定为一种pp - GalNAc - T家族基因。然而，GALNTL5没有糖基转移酶活性。在小鼠中，Galntl5的表达仅限于分化中的精子细胞，单倍剂量不足会导致精子蛋白质组成异常且无法游动。此外，在被诊断为弱精子症（精子活力低）的患者中检测到人类GALNTL5的杂合缺失。尽管这些发现表明GALNTL5是哺乳动物成熟精子形成所必需的功能分子，但GALNTL5在精子发生中的具体功能仍不清楚。为了阐明这一作用，我们建立了小鼠精母细胞系GC - 2spd(ts)，该细胞系表现出药物诱导的GALNTL5表达。有趣的是，在所得细胞系中持续表达GALNTL5会导致细胞凋亡并伴有细胞收缩，GALNTL5定位于内质网（ER），并与两种内质网驻留伴侣蛋白钙连蛋白和BiP（GRP78）相关联。钙连蛋白识别并强烈结合在内质网中修饰的GALNTL5分子上的N - 聚糖。相反，内质网驻留的BiP可能与GALNL5结合，而不考虑其糖基化状态。GALNTL5的表达消除了钙连蛋白与错误折叠的底物蛋白之间的结合，表明GALNTL5直接阻断了钙连蛋白的功能。此外，GALNTL5与钙连蛋白之间的相互作用降低了BiP蛋白的水平，因此也降低了内质网（ER）、高尔基体和细胞质中驻留蛋白的表达水平。使用小干扰RNA敲低GC - 2spd(ts)细胞系中钙连蛋白或BiP的功能，证实了这些蛋白水平的降低。此外，持续表达GALNTL5导致细胞结构变化，包括顺式高尔基体的位置以及内质网网络的改变。这些结果强烈表明，GALNTL5通过阻断内质网功能，促进了分化中的精子细胞特有的细胞结构改变。
