Structural basis for matriglycan synthesis by the LARGE1 dual glycosyltransferase

Abstract

LARGE1 is a bifunctional glycosyltransferase responsible for generating a long linear polysaccharide termed matriglycan that links the cytoskeleton and the extracellular matrix and is required for proper muscle function. This matriglycan polymer is made with an alternating pattern of xylose and glucuronic acid monomers. Mutations in the LARGE1 gene have been shown to cause life-threatening dystroglycanopathies through the inhibition of matriglycan synthesis. Despite its major role in muscle maintenance, the structure of the LARGE1 enzyme and how it assembles in the Golgi are unknown. Here we present the structure of LARGE1, obtained by a combination of X-ray crystallography and single-particle cryo-EM. We found that LARGE1 homo-dimerizes in a configuration that is dictated by its coiled-coil stem domain. The structure shows that this enzyme has two canonical GT-A folds within each of its catalytic domains. In the context of its dimeric structure, the two types of catalytic domains are brought into close proximity from opposing monomers to allow efficient shuttling of the substrates between the two domains. Together, with putative retention of matriglycan by electrostatic interactions, this dimeric organization offers a possible mechanism for the ability of LARGE1 to synthesize long matriglycan chains. The structural information further reveals the mechanisms in which disease-causing mutations disrupt the activity of LARGE1. Collectively, these data shed light on how matriglycan is synthesized alongside the functional significance of glycosyltransferase oligomerization.

The underlying mechanisms of arenaviral entry through matriglycan

“…This enzyme contains a cytoplasmic/transmembrane region, a coiled-coil (stem) domain, a xylosyltransferase (Xyl-T) domain, a linker region, and a glucuronyltransferase (GlcA-T) domain ( Figure 1 ). The ectodomain structure of LARGE1 (residues 29–756) ( Peyrard et al, 1999 ) was recently solved ( Joseph et al, 2022 ; Katz and Diskin, 2022 ), providing insights into how matriglycan is synthesized. This enzyme exists as a homodimer, with two copies of each catalytic domain.…”

“…This enzyme exists as a homodimer, with two copies of each catalytic domain. Both catalytic domains consist of a canonical GT-A fold that contains an α/β/α sandwich, similar to a Rossman fold for nucleotide-binding proteins ( Harrus et al, 2018 ; Taujale et al, 2020 ; Joseph et al, 2022 ; Katz and Diskin, 2022 ). Furthermore, the catalytic domains both contain a canonical ‘DXD’ motif, which forms part of each active site.…”

“…The DXD motif of each domain straddles between the major and minor β-sheets where it engages with incoming monosaccharide-UDP substrates. The Xyl-T domain, a retaining transferase member of the GT 8 family ( Drula et al, 2022 ), contains a DXD motif with a bound Mn 2+ ion that facilitates the addition of a Xyl residue to the growing matriglycan chain ( Katz and Diskin, 2022 ). The GlcA-T domain, an inverting transferase member of the GT 49 family ( Drula et al, 2022 ), is predicted to coordinate a Mn 2+ ion (or another divalent ion) in its DXD domain.…”

“…In this form, two LARGE1 monomers are associated such that each catalytic domain interacts with its cognate domain on the second monomer ( Figure 1 ). Oligomerization in a parallel-dimeric form was found to be dependent upon the presence of the coiled-coil (CC) domain ( Katz and Diskin, 2022 ). On each monomer, the two catalytic pockets face in opposite directions.…”

“…Interestingly, several positively charged patches exist along the perimeter of the enzyme. These areas likely facilitate the growing (negatively charged) matriglycan to wrap around LARGE1 while having its non-reducing end stay near the two active sites ( Katz and Diskin, 2022 ). Furthermore, LARGE1 synthesizes matriglycan processively, rather than distributively, through the use of these adjacent catalytic domains ( Figure 1 ).…”