ABSTRACT
Neutralizing antibodies have become an important tool in treating infectious diseases. Recently, two separate approaches yielded successful antibody treatments for Ebola-one from genetically humanized mice and the other from a human survivor. Here, we describe parallel efforts using both humanized mice and convalescent patients to generate antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, which yielded a large collection of fully human antibodies that were characterized for binding, neutralization, and three-dimensional structure. On the basis of these criteria, we selected pairs of highly potent individual antibodies that simultaneously bind the receptor binding domain of the spike protein, thereby providing ideal partners for a therapeutic antibody cocktail that aims to decrease the potential for virus escape mutants that might arise in response to selective pressure from a single-antibody treatment.
Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Betacoronavirus/immunology , Coronavirus Infections/immunology , Pneumonia, Viral/immunology , Spike Glycoprotein, Coronavirus/immunology , Adolescent , Adult , Angiotensin-Converting Enzyme 2 , Animals , Antibodies, Neutralizing/chemistry , Antibodies, Viral/chemistry , Antibody Affinity , Antibody-Dependent Cell Cytotoxicity , Betacoronavirus/chemistry , Binding Sites, Antibody , Broadly Neutralizing Antibodies/chemistry , Broadly Neutralizing Antibodies/immunology , COVID-19 , Cell Line , Coronavirus Infections/therapy , Cytophagocytosis , Epitopes , Humans , Immunization, Passive , Mice , Middle Aged , Models, Molecular , Neutralization Tests , Pandemics , Peptidyl-Dipeptidase A/metabolism , Protein Interaction Domains and Motifs , Receptors, Coronavirus , Receptors, Virus/metabolism , Severe acute respiratory syndrome-related coronavirus/immunology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Young Adult , COVID-19 SerotherapyABSTRACT
MuSK, a muscle-specific receptor tyrosine kinase that is activated by agrin, has a critical role in neuromuscular synapse formation. In cultured myotubes, agrin stimulates the rapid phosphorylation of MuSK, leading to MuSK activation and tyrosine phosphorylation and clustering of acetylcholine receptors. Agrin, however, fails to stimulate tyrosine phosphorylation of MuSK that is force-expressed in myoblasts and fibroblasts, indicating that myotubes contain an additional activity that is required for agrin to stimulate MuSK. Certain glycosyltransferases are expressed selectively at synaptic sites in skeletal muscle, raising the possibility that carbohydrate modifications of MuSK, catalyzed by glycosyltransferases expressed selectively in myotubes, may be essential for agrin to bind and activate MuSK. We identifed two N-linked glycosylation sites in MuSK, and we expressed MuSK mutants lacking one or both N-linked sites into MuSK mutant myotubes to determine whether N-linked carbohydrate modifications of MuSK have a role in MuSK activation. We found that N-linked glycosylation restrains ligand-independent tyrosine phosphorylation of MuSK and downstream signaling but is not necessary for agrin to stimulate MuSK.
Subject(s)
Muscle, Skeletal/physiology , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Cholinergic/metabolism , Receptors, Cholinergic/physiology , Amino Acid Substitution , Animals , Base Sequence , Binding Sites , Cell Line , Conserved Sequence , DNA Primers , Electric Organ/physiology , Enzyme Activation , Glycosylation , Humans , Lectins/metabolism , Mutagenesis, Site-Directed , Phosphorylation , Protein Processing, Post-Translational , Receptor Protein-Tyrosine Kinases/chemistry , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Cholinergic/chemistry , Receptors, Cholinergic/genetics , Recombinant Proteins/metabolism , Torpedo , TransfectionABSTRACT
Muscle-specific kinase (MuSK) is a receptor tyrosine kinase expressed selectively in skeletal muscle. During neuromuscular synapse formation, agrin released from motor neurons stimulates MuSK autophosphorylation in the kinase activation loop and in the juxtamembrane region, leading to clustering of acetylcholine receptors. We have determined the crystal structure of the cytoplasmic domain of unphosphorylated MuSK at 2.05 A resolution. The structure reveals an autoinhibited kinase domain in which the activation loop obstructs ATP and substrate binding. Steady-state kinetic analysis demonstrates that autophosphorylation results in a 200-fold increase in k(cat) and a 10-fold decrease in the K(m) for ATP. These studies provide a molecular basis for understanding the regulation of MuSK catalytic activity and suggest that an additional in vivo component may contribute to regulation via the juxtamembrane region.