de Las Rivas Matilde, Paul Daniel Earnest James, Coelho Helena, Lira-Navarrete Erandi, Raich Lluis, Compañón Ismael, Diniz Ana, Lagartera Laura, Jiménez-Barbero Jesús, Clausen Henrik, Rovira Carme, Marcelo Filipa, Corzana Francisco, Gerken Thomas A, Hurtado-Guerrero Ramon
BIFI, University of Zaragoza, BIFI-IQFR (CSIC) Joint Unit, Mariano Esquillor s/n, Campus Rio Ebro, Edificio I+D, Zaragoza 50018, Spain.
Departments of Biochemistry, Pediatrics and Chemistry, Case Western Reserve University, Cleveland, Ohio 44106, United States.
ACS Cent Sci. 2018 Sep 26;4(9):1274-1290. doi: 10.1021/acscentsci.8b00488. Epub 2018 Sep 14.
Mucin-type -glycosylation is initiated by a family of polypeptide GalNAc-transferases (GalNAc-Ts) which are type-II transmembrane proteins that contain Golgi luminal catalytic and lectin domains that are connected by a flexible linker. Several GalNAc-Ts, including GalNAc-T4, show both long-range and short-range prior glycosylation specificity, governed by their lectin and catalytic domains, respectively. While the mechanism of the lectin-domain-dependent glycosylation is well-known, the molecular basis for the catalytic-domain-dependent glycosylation of glycopeptides is unclear. Herein, we report the crystal structure of GalNAc-T4 bound to the diglycopeptide GATGAGAGAGTTPGPG (containing two α-GalNAc glycosylated Thr (T*), the PXP motif and a "naked" Thr acceptor site) that describes its catalytic domain glycopeptide GalNAc binding site. Kinetic studies of wild-type and GalNAc binding site mutant enzymes show the lectin domain GalNAc binding activity dominates over the catalytic domain GalNAc binding activity and that these activities can be independently eliminated. Surprisingly, a flexible loop protruding from the lectin domain was found essential for the optimal activity of the catalytic domain. This work provides the first structural basis for the short-range glycosylation preferences of a GalNAc-T.
粘蛋白型O-糖基化由一组多肽N-乙酰半乳糖胺转移酶（GalNAc-Ts）起始，这些酶是II型跨膜蛋白，包含通过柔性接头连接的高尔基体腔催化结构域和凝集素结构域。几种GalNAc-Ts，包括GalNAc-T4，分别显示出由其凝集素和催化结构域控制的长程和短程优先糖基化特异性。虽然凝集素结构域依赖性糖基化的机制是众所周知的，但糖肽催化结构域依赖性糖基化的分子基础尚不清楚。在此，我们报道了GalNAc-T4与二糖肽GATGAGAGAGTTPGPG（含有两个α-半乳糖胺糖基化苏氨酸（T*）、PXP基序和一个“裸”苏氨酸受体位点）结合的晶体结构，该结构描述了其催化结构域糖肽N-乙酰半乳糖胺结合位点。野生型和N-乙酰半乳糖胺结合位点突变酶的动力学研究表明，凝集素结构域N-乙酰半乳糖胺结合活性优于催化结构域N-乙酰半乳糖胺结合活性，并且这些活性可以独立消除。令人惊讶的是，发现从凝集素结构域突出的一个柔性环对于催化结构域的最佳活性至关重要。这项工作为GalNAc-T的短程糖基化偏好提供了第一个结构基础。
