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Background: α-dystroglycanopathies are congenital muscular dystrophies in which genetic mutations cause the decrease or absence of a unique and complex O-linked glycan called matriglycan. This hypoglycosylation of O-linked matriglycan on the α-dystroglycan (α-DG) protein subunit abolishes or reduces the protein binding to extracellular ligands such as laminins in skeletal muscles, leading to compromised survival of muscle cells after contraction. Methods: Surrogate molecular linkers reconnecting laminin-211 and the dystroglycan β-subunit through bispecific antibodies can be engineered to improve muscle function in the α-dystroglycanopathies. This study reports the metabolic engineering of a novel glycofusion bispecific (GBi) antibody that fuses the mucin-like domain of the α-DG to the light chain of an anti-β-DG subunit antibody. Results: Transient HEK production with the co-transfection of LARGE1, the glycoenzyme responsible for the matriglycan modification, produced the GBi antibody only with a light matriglycan modification and a weak laminin-211 binding activity. However, when a sugar feed mixture of uridine, galactose, and manganese ion (Mn2+) was added to the culture medium, the GBi antibody produced exhibited a dramatically enhanced matriglycan modification and a much stronger laminin-binding activity. Conclusions: Further investigation has revealed that Mn2+ in the sugar feeds played a critical role in increasing the matriglycan modification of the GBi antibody, key for the function of the resulting bispecific antibody.

The diffusion of viruses at the cell membrane is essential to reach a suitable entry site and initiate subsequent internalization. Although many viruses take advantage of glycosaminoglycans (GAG) to bind to the cell surface, little is known about the dynamics of the virus–GAG interactions. Here, single-particle tracking of the initial interaction of individual herpes simplex virus 1 (HSV-1) virions reveals a heterogeneous diffusive behavior, regulated by cell-surface GAGs with two main diffusion types: confined and normal free. This study reports that different GAGs can have competing influences in mediating diffusion on the cells used here: chondroitin sulfate (CS) enhances free diffusion but hinders virus attachment to cell surfaces, while heparan sulfate (HS) promotes virus confinement and increases entry efficiency. In addition, the role that the viral mucin-like domains (MLD) of the HSV-1 glycoprotein C plays in facilitating the diffusion of the virus and accelerating virus penetration into cells is demonstrated. Together, our results shed new light on the mechanisms of GAG-regulated virus diffusion at the cell surface for optimal internalization. These findings may be extendable to other GAG-binding viruses.

The homing of lymphocytes from blood to gut-associated lymphoid tissue is regulated by interaction between integrin α4β7 with mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) expressed on the endothelium of high endothelial venules (HEVs). However, the molecular basis of mucin-like domain, a specific structure of MAdCAM-1 regulating integrin α4β7-mediated cell adhesion remains obscure. In this study, we used heparan sulfate (HS), which is a highly acidic linear polysaccharide with a highly variable structure, to mimic the negative charges of the extracellular microenvironment and detected the adhesive behaviors of integrin α4β7 expressing 293T cells to immobilized MAdCAM-1 in vitro. The results showed that HS on the surface significantly promoted integrin α4β7-mediated cell adhesion, decreased the percentage of cells firmly bound and increased the rolling velocities at high wall shear stresses, which was dependent on the mucin-like domain of MAdCAM-1. Moreover, breaking the negative charges of the extracellular microenvironment of CHO-K1 cells expressing MAdCAM-1 with sialidase inhibited cell adhesion and rolling velocity of 293T cells. Mechanistically, electrostatic repulsion between mucin-like domain and negative charges of the extracellular microenvironment led to a more upright conformation of MAdCAM-1, which facilitates integrin α4β7-mediated cell adhesion. Our findings elucidated the important role of the mucin-like domain in regulating integrin α4β7-mediated cell adhesion, which could be applied to modulate lymphocyte homing to lymphoid tissues or inflammatory sites.

Ebola virus (EBOV) is the cause of sporadic outbreaks of human hemorrhagic disease in Africa, and the best-characterized virus in the filovirus family. The West African epidemic accelerated the clinical development of vaccines and therapeutics, leading to licensure of vaccines and antibody-based therapeutics for human use in recent years. The most widely used vaccine is based on vesicular stomatitis virus (VSV) expressing the EBOV glycoprotein (GP) (VSV-EBOV). Due to its favorable immune cell targeting, this vaccine has also been used as a base vector for the development of second generation VSV-based vaccines against Influenza, Nipah, and Zika viruses. However, in these situations, it may be beneficial if the immunogenicity against EBOV GP is minimized to induce a better protective immune response against the other foreign immunogen. Here, we analyzed if EBOV GP can be truncated to be less immunogenic, yet still able to drive replication of the vaccine vector. We found that the EBOV GP glycan cap and the mucin-like domain are both dispensable for VSV-EBOV replication. The glycan cap, however, appears critical for mediating a protective immune response against lethal EBOV challenge in mice.

Jingmen viruses are newly described segmented flavi-like viruses that have a worldwide distribution in ticks and have been associated with febrile illnesses in humans. Computational analyses were used to predict that Jingmen flavi-like virus glycoproteins have structural features of class II viral fusion proteins, including an ectodomain consisting of beta-sheets and short alpha-helices, a fusion peptide with interfacial hydrophobicity and a three-domain architecture. Jingmen flavi-like virus glycoproteins have a sequence enriched in serine, threonine, and proline at the amino terminus, which is a feature of mucin-like domains. Several of the serines and threonines are predicted be modified by the addition of O-linked glycans. Some of the glycoproteins are predicted to have an additional mucin-like domain located prior to the transmembrane anchor, whereas others are predicted to have a stem consisting of two alpha-helices. The flavivirus envelope protein and Jingmen flavi-virus glycoproteins may have diverged from a common class II precursor glycoprotein with a mucin-like domain or domains acquired after divergence.

Abstract Background The genus Ebolavirus includes five distinct viruses. Four of these viruses cause hemorrhagic fever in humans. Currently there are no licensed vaccines for any of them; however, several vaccines are under development. Ebola virus envelope glycoprotein (GP1,2) is highly immunogenic, but antibodies frequently arise against its least conserved mucin-like domain (MLD). We hypothesized that immunization with MLD-deleted GP1,2 (GPΔMLD) would induce cross-species immunity by making more conserved regions accessible to the immune system. Methods To test this hypothesis, mice were immunized with retrovirus-like particles (retroVLPs) bearing Ebola virus GPΔMLD, DNA plasmids (plasmo-retroVLP) that can produce such retroVLPs in vivo, or plasmo-retroVLP followed by retroVLPs. Results Cross-species neutralizing antibody and GP1,2-specific cellular immune responses were successfully induced. Conclusion Our findings suggest that GPΔMLD presented through retroVLPs may provide a strategy for development of a vaccine against multiple ebolaviruses. Similar vaccination strategies may be adopted for other viruses whose envelope proteins contain highly variable regions that may mask more conserved domains from the immune system.