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

Alpha-Dystroglycan (alpha-DG) is uniquely modified on O-mannose sites by a repeating disaccharide (-Xylalpha1,3-GlcAbeta1,3-)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 alpha-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.

Identification of residues in Lassa virus glycoprotein 1 involved in receptor switch

Lassa virus (LASV) is an enveloped, negative-sense RNA virus that causes Lassa hemorrhagic fever, for which there are limited treatment options. Successful LASV entry requires the viral glycoprotein 1 (GP1) to undergo a receptor switch from its primary receptor alpha-dystroglycan (α-DG) to its endosomal receptor lysosome-associated membrane protein 1 (LAMP1). A conserved histidine triad in LASV GP1 has been reported to be responsible for receptor switch. To test the hypothesis that other non-conserved residues also contribute to receptor switch, we constructed a series of GP1 mutant proteins and tested them for binding to LAMP1. Four residues, L84, K88, L107, and H170, were identified as critical for receptor switch. Substituting any of the four residues with the corresponding lymphocytic choriomeningitis virus residue (L84N, K88E, L10F, and H170S) reduced the binding affinity of GP1LASV for LAMP1. Moreover, all the mutations caused decreases in GPC-mediated membrane fusion at both pH 4.5 and 5.2. The infectivity of pseudotyped viruses bearing either GPCL84N or GPCK88E decreased sharply in multiple cell types, whereas L107F and H170S had only mild effects on infectivity. Notably, in LAMP1 knockout cells, all four mutants showed reduced pseudovirus infectivity. Using biolayer light interferometry assay, we found that all four mutants had decreased binding affinity to LAMP1, in the order L84N > L107F > K88E > H170S.

Contrasting the Conformational Effects of α-O-GalNAc and α-O-Man Glycan Protein Modifications and their Impact on the Mucin-Like Region of alpha-Dystroglycan

We have carried out a comparative study of the conformational impact of modifications to threonine residues of either α-O-Man or α-O-GalNAc in the context of a sequence from the mucin-like region of α-dystroglycan. Both such modifications can coexist in this domain of the glycoprotein. Solution NMR experiments and molecular dynamics calculations were employed. Comparing the results for a unmodified peptide Ac- PPTTTTKKP-NH2 sequence from α-dystroglycan, and glycoconjugates with either modification on the Ts, we find that the impact of the α-O-Man modification on the peptide scaffold is quite limited, while that of the α-O-GalNAc is more profound. The results for the α-O-GalNAc glycoconjugate are consistent with what has been seen earlier in other systems. Further examination of the NMR-based structure and the MD results suggest a more extensive network of hydrogen bond interactions within the α-O-GalNAc-threonine residue than has been previously appreciated, which influence the properties of the protein backbone. The conformational effects are relevant to the mechanical properties of α-dystroglycan.

TRAPPC11-related muscular dystrophy with hypoglycosylation of alpha-dystroglycan in skeletal muscle and brain.

Background: TRAPPC11, a subunit of the transport protein particle (TRAPP) complex is important for complex integrity and anterograde membrane transport from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment. Several individuals with TRAPPC11 mutations have been reported with muscle weakness and other features including brain, liver, skeletal and eye involvement. A detailed analysis of brain and muscle biopsies will further our understanding of the presentation and etiology of TRAPPC11-disease. Methods: We describe five cases of early-onset TRAPPC11–related muscular dystrophy with a systematic review of muscle pathology in all five individuals, post-mortem brain pathology findings in one individual, and membrane trafficking assays in another. Results: All affected individuals presented in infancy with muscle weakness, motor delay and elevated serum creatine kinase (CK). Additional features included cataracts, liver disease, intellectual disability, cardiomyopathy, movement disorder, and structural brain abnormalities. Muscle pathology in all five revealed dystrophic changes, universal hypoglycosylation of alpha-dystroglycan and variably reduced dystrophin-associated complex proteins. Membrane trafficking assays showed defective Golgi trafficking in one individual. Neuropathological examination of one individual revealed cerebellar atrophy, granule cell hypoplasia, Purkinje cell (PC) loss and dendritic neurodegeneration, reduced alpha-dystroglycan (IIH6) expression in PC and dentate neurons, and absence of neuronal migration defects. Conclusions: This report suggests that recessive mutations in TRAPPC11 are linked to muscular dystrophies with hypoglycosylation of alpha-dystroglycan. The structural brain involvement that we document for the first time resembles the pathology previously reported in N-linked congenital disorders of glycosylation (CDG) such as PMM2-CDG, suggesting defects in multiple glycosylation pathways in this condition.

TRAPPC11-Related Muscular Dystrophy with Hypoglycosylation of Alpha-Dystroglycan in Skeletal Muscle and Brain

Background: TRAPPC11, a subunit of the transport protein particle (TRAPP) complex is important for complex integrity and anterograde membrane transport from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment. Several individuals with TRAPPC11 mutations have been reported with muscle weakness and other features including brain, liver, skeletal and eye involvement. A detailed analysis of brain and muscle biopsies will further our understanding of the presentation and etiology of TRAPPC11-disease.Methods: We describe five cases of early-onset TRAPPC11–related muscular dystrophy with a systematic review of muscle pathology and post-mortem brain pathology findings in one individual, and membrane trafficking assays in another.Results: All affected individuals presented in infancy with muscle weakness, motor delay and elevated serum creatine kinase (CK). Additional features included cataracts, liver disease, intellectual disability, cardiomyopathy, movement disorder, and structural brain abnormalities. Muscle pathology in all five revealed dystrophic changes, universal hypoglycosylation of alpha-dystroglycan and variably reduced dystrophin-associated complex proteins. Membrane trafficking assays showed defective Golgi trafficking in one individual. Neuropathological examination of one individual revealed cerebellar atrophy, granule cell hypoplasia, Purkinje cell (PC) loss and dendritic neurodegeneration, reduced alpha-dystroglycan (IIH6) expression in PC and dentate neurons, and absence of neuronal migration defects.Conclusions: This report suggests that recessive mutations in TRAPPC11 are linked to muscular dystrophies with hypoglycosylation of alpha-dystroglycan. The structural brain involvement that we document for the first time resembles the pathology previously reported in N-linked congenital disorders of glycosylation (CDG) such as PMM2-CDG, suggesting defects in multiple glycosylation pathways in this condition.

N-glycosylated SGK196 suppresses the metastasis of basal-like breast cancer cells

SGK196 is a protein O-mannose kinase involved in an indispensable phosphorylation step during laminin-binding glycan synthesis on alpha-dystroglycan (α-DG). However, the function of SGK196 in cancer diseases remains elusive. In the current study, we demonstrated that SGK196 is primarily modified by N-glycosylation in breast cancer (BC) cells. Furthermore, gain and loss-of-function studies showed that N-glycosylated SGK196 suppresses cell migration, invasion, and metastasis in BC, particularly in the basal-like breast cancer (BLBC) type. In addition, we found that SGK196 N-glycosylation performs the regulatory function through the PI3K/AKT/GSK3β signaling pathway. Collectively, our results show that N-glycosylated SGK196 plays suppression roles in BLBC metastases, therefore providing new insights into SGK196 function in BC.