Biology subjects

Campbell, K. P.

Publications and source records attributed to Campbell, K. P..

7 recordsLinked to original sources

bioRxiv2025

Xylose phosphatase activity of dystroglycan self-regulates its receptor function

Dystroglycan (DG), a transmembrane receptor crucial for tissue development and pathogen entry, harbors a glycan composed of xylose and glucuronic acid called matriglycan. Loss of matriglycan or reduction in its length affects DG function, causing dystroglycanopathies. However, the mechanism underlying matriglycan extension is unknown. Here, we show that a Golgi xylose kinase facilitates initiation of matriglycan synthesis by transiently adding a phosphate to the xylose of matriglycan primer. Matriglycan extends when the phosphate is removed from xylose by the N-terminal domain of dystroglycan (DGN). DGN has the DXDXT/V motif found in haloacid dehalogenase (HAD) domains of hydrolases, conditional mutations in which reduce matriglycan length and cause disease in mice. Our work reveals an unexpected glycan phosphatase function of DG in regulating matriglycan extension on itself.

biochemistry↗

bioRxiv2023

Identification of a short, single site matriglycan that maintains neuromuscular function in the mouse

Matriglycan (-1,3-{beta}-glucuronic acid-1,3--xylose-) is a polysaccharide that is synthesized on -dystroglycan, where it functions as a high-affinity glycan receptor for extracellular proteins, such as laminin, perlecan and agrin, thus anchoring the plasma membrane to the extracellular matrix. This biological activity is closely associated with the size of matriglycan. Using high-resolution mass spectrometry and site-specific mutant mice, we show for the first time that matriglycan on the T317/T319 and T379 sites of -dystroglycan are not identical. T379-linked matriglycan is shorter than the previously characterized T317/T319-linked matriglycan, although it maintains its laminin binding capacity. Transgenic mice with only the shorter T379-linked matriglycan exhibited mild embryonic lethality, but those that survived were healthy. The shorter T379-linked matriglycan exists in multiple tissues and maintains neuromuscular function in adult mice. In addition, the genetic transfer of -dystroglycan carrying just the short matriglycan restored grip strength and protected skeletal muscle from eccentric contraction-induced damage in muscle-specific dystroglycan knock-out mice. Due to the effects that matriglycan imparts on the extracellular proteome and its ability to modulate cell-matrix interactions, our work suggests that differential regulation of matriglycan length in various tissues optimizes the extracellular environment for unique cell types.

biochemistry↗

bioRxiv2022

Dystroglycan N-terminal domain enables LARGE1 to extend matriglycan on α-dystroglycan and prevents muscular dystrophy

Dystroglycan (DG) requires extensive post-translational processing to function as a receptor for extracellular matrix proteins containing laminin-G-like (LG) domains. Matriglycan is an elongated polysaccharide of alternating xylose and glucuronic acid that is uniquely synthesized on -dystroglycan (-DG) by like-acetylglucosaminyltransferase-1 (LARGE1) and binds with high affinity to matrix proteins like laminin. Defects in the post-translational processing of -DG that result in a shorter form of matriglycan reduce the size of -DG and decrease laminin binding, leading to various forms of muscular dystrophy. However, little is known regarding mechanisms that generate full-length matriglycan on -DG (~150-250 kDa). Here, we show that LARGE1 can only synthesize a short, non-elongated form of matriglycan in mouse skeletal muscle that lacks the DG N-terminus (-DGN), resulting in a ~100-125 kDa -DG. This smaller form of -DG binds laminin and maintains specific force but does not prevent muscle pathophysiology, including reduced force induced by eccentric contractions and abnormalities in neuromuscular junctions. Collectively, our study demonstrates that -DGN is required for LARGE1 to extend matriglycan to its full mature length on -DG and thus prevent muscle pathophysiology.

biochemistry↗

bioRxiv2022

Structure and mechanism of LARGE1 matriglycan polymerase

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.

biochemistry↗

bioRxiv2021

Large1 Gene Transfer in Older myd Mice with Severe Muscular Dystrophy Restores Muscle Function and Greatly Improves Survival

Muscular dystrophy is a progressive and ultimately lethal neuromuscular disease due to lack of therapeutic options that restore muscle function. Gene editing and gene transfer hold great promise as therapies for various neuromuscular diseases when administered prior to the onset of severe clinical symptoms. However, the efficacy of these strategies for restoring neuromuscular function and improving survival in the late stages of muscular dystrophy with severe muscle pathophysiology is unknown. Dystroglycanopathies are muscular dystrophies characterized by extensive skeletal muscle degeneration and, in many cases, are accompanied by eye and brain abnormalities. Thus far, mutations in at least eighteen human genes are known to cause dystroglycanopathies, including those in the like-acetylglucosaminyltransferase-1 (LARGE1) gene. LARGE1 encodes a xylosyl- and glucuronosyltransferase that modifies -dystroglycan (-DG) with matriglycan, a linear repeating disaccharide of alternating xylose and glucuronic acid that binds to the laminin G-like domains of extracellular matrix proteins with high affinity. Largemyd/Largemyd (myd) mice lack expression of Large1, and exhibit severe skeletal muscle pathophysiology, impaired mobility, and a drastically reduced lifespan (50% survivorship at 35 weeks of age). Here, we show that systemic delivery of AAV2/9 CMV Large1 (AAVLarge1) in >34-week-old myd mice with advanced disease restores matriglycan expression, attenuates skeletal muscle pathophysiology, improves motor and respiratory function, and normalizes systemic metabolism, which collectively and dramatically extends survival. Our results demonstrate that in a mouse model of muscular dystrophy, skeletal muscle function can be restored, illustrating its remarkable plasticity, and that survival can be greatly improved even after the onset of severe skeletal muscle pathophysiology.

physiology↗

bioRxiv2021

Cell Surface Glycan Engineering Reveals that Matriglycan Alone can Recapitulate Dystroglycan Binding and Function

-Dystroglycan (-DG) is uniquely modified on O-mannose sites by a repeating disaccharide (-Xyl1,3-GlcA{beta}1,3-)n termed matriglycan, which is a receptor for laminin-G domain-containing proteins and employed by old-world arenaviruses for infection. Using chemoenzymatically synthesized matriglycans printed as a microarray, we demonstrated length-dependent binding to Laminin, Lassa virus GP1, and the clinically-important antibody IIH6. Utilizing an enzymatic engineering approach, an N-linked glycoprotein was converted into a IIH6-positive Laminin-binding glycoprotein. Engineering of the surface of cells deficient for either -DG or O-mannosylation with matriglycans of sufficient length recovered infection with a Lassa-pseudovirus. Finally free matriglycan in a dose and length dependent manner inhibited viral infection of wildtype cells. These results indicate that matriglycan alone is necessary and sufficient for IIH6 staining, Laminin and LASV GP1 binding, and Lassa-pseudovirus infection and support a model in which it is a tunable receptor for which increasing chain length enhances ligand-binding capacity. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY Graphical Abstract C_FIG_DISPLAY

biochemistry↗

bioRxiv2020

POMK Regulates Dystroglycan Function via LARGE-mediated Elongation of Matriglycan

Matriglycan [-GlcA-{beta}1,3-Xyl-1,3-]n serves as a scaffold in many tissues for extracellular matrix proteins containing laminin-G domains including laminin, agrin, and perlecan. Like-acetylglucosaminyltransferase-1 (LARGE) synthesizes and extends matriglycan on -dystroglycan (-DG) during skeletal muscle differentiation and regeneration; however, the mechanisms which regulate matriglycan elongation are unknown. Here, we show that Protein O-Mannose Kinase (POMK), which phosphorylates mannose of core M3 (GalNac-{beta}1,3-GlcNac-{beta}1,4-Man) preceding matriglycan synthesis, is required for LARGE-mediated generation of full-length matriglycan on -DG ([~]150 kDa). In the absence of POMK, LARGE synthesizes a very short matriglycan resulting in a [~]90 kDa -DG in mouse skeletal muscle which binds laminin but cannot prevent eccentric contraction-induced force loss or muscle pathology. Solution NMR spectroscopy studies demonstrate that LARGE directly interacts with core M3 and binds preferentially to the phosphorylated form. Collectively, our study demonstrates that phosphorylation of core M3 by POMK enables LARGE to elongate matriglycan on -DG, thereby preventing muscular dystrophy.

biochemistry↗