Structure and mechanism of LARGE1 matriglycan polymerase

Abstract

Matriglycan is a linear polysaccharide of alternating xylose and glucuronate that binds extracellular matrix proteins and acts as a receptor for Lassa fever virus. LARGE1 synthesizes matriglycan on dystroglycan and mutations in LARGE1 cause muscular dystrophy with abnormal brain development. However, the mechanism of matriglycan polymerization by LARGE1 is unknown. Here, we report the cryo-EM structure of LARGE1. We show that LARGE1 functions as a dimer to polymerize matriglycan by alternating activities between the xylose transferase domain on one protomer and the glucuronate transferase domain on the other protomer. Biochemical analyses using a recombinant Golgi form of dystroglycan reveal that LARGE1 polymerizes matriglycan processively. Our results provide mechanistic insights into LARGE1 function and may facilitate novel therapeutic strategies for treating neuromuscular disorders or arenaviral infections.One-Sentence SummaryDimeric LARGE1 processively polymerizes matriglycan on dystroglycan using orthogonal active sites on alternate protomers.

Deep Mutational Scanning in Disease-related Genes with Saturation Mutagenesis-Reinforced Functional Assays (SMuRF)

“…Interestingly, SMuRF also showed that the variants in the XylT domain tend to be more disruptive than those in the GlcAT domain (p-values<2.22e-16). A previous IIH6C4 western blot experiment revealed a similar observation, demonstrating that mutations deactivating the GlcAT domain, but not the XylT domain, can generate a faint band indicative of glycosylated matriglycan 79 (Supplementary Discussion 6). Again, the differences in SMuRF scores between domains were observed exclusively in missense variants and not in synonymous variants (p-values>0.05) (Fig.…”

“…α-DG glycosylation plays a crucial role in LASV viral entry. It is possible that certain variants, despite conferring lower enzymatic activity for α-DG glycosylation, are favored by selection in populations where LASV is epidemic 79,97 . These aspects emphasize the need for orthogonal validation assays. In our study, we created myogenic platform cell lines to validate the findings from the flow-cytometry assay.…”

“…LARGE1 has two catalytic domains: a xylose transferase (XylT) domain (p.138-413) and a glucuronate transferase (GlcAT) domain (p.414-756) 79 . They are each responsible for adding one unit of the polysaccharide matriglycan chain, which consists of alternating xylose and glucuronate units.…”

Structural basis for matriglycan synthesis by the LARGE1 dual glycosyltransferase

“…This proximity between the two active sites increases the probability that the growing matriglycan chain will be sequentially modified by the two domains before the matriglycan polymer dissociates from LARGE1. While preparing this manuscript for submission, a complementary investigation of LARGE1 was deposited in a preprint server [ 62 ]. This work also supports the notion that active sites from two opposite chains contribute for the synthesis of matriglycan, which likely represent the most efficient way that LARGE1 can generate matriglycan.…”

Synthesis of the matriglycan hexasaccharide, -3Xylα1-3GlcAβ1-trimer and its interaction with laminin

“…6 Joseph et al recently advocated the extending mechanism of matriglycan on LARGE1. 11 Matriglycan also exhibits high affinity to Lassa virus glycoprotein 1 (LASV GP1). 12,13 Boons et al chemo-enzymatically obtained a matriglycan oligosaccharide in 2022.…”