SUMMARY Compelling evidence indicates that the CRISPR-Cas system protects prokaryotes from viruses and other potential genome invaders. This adaptive prokaryotic immune system arises from the clustered regularly interspaced short palindromic repeats (CRISPRs) found in prokaryotic genomes, which harbor short invader-derived sequences, and the CRISPR-associated (Cas) protein-coding genes. Here we have identified a CRISPR-Cas effector complex that is comprised of small invader-targeting RNAs from the CRISPR loci (termed prokaryotic silencing (psi)RNAs) and the RAMP module (or Cmr) Cas proteins. The psiRNA-Cmr protein complexes cleave complementary target RNAs at a fixed distance from the 3' end of the integral psiRNAs. In Pyrococcus furiosus, psiRNAs occur in two size forms that share a common 5' sequence tag but have distinct 3' ends that direct cleavage of a given target RNA at two distinct sites. Our results indicate that prokaryotes possess a unique RNA silencing system that functions by homology-dependent cleavage of invader RNAs.

The dynamic glycosylation of serine or threonine residues on nuclear and cytosolic proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc) is abundant in all multicellular eukaryotes. On several proteins, O-GlcNAc and O-phosphate alternatively occupy the same or adjacent sites, leading to the hypothesis that one function of this saccharide is to transiently block phosphorylation. The diversity of proteins modified by O-GlcNAc implies its importance in many basic cellular and disease processes. Here we systematically examine the current data implicating O-GlcNAc as a regulatory modification important to signal transduction cascades.

Summary The SARS-CoV-2 betacoronavirus uses its highly glycosylated trimeric Spike protein to bind to the cell surface receptor angiotensin converting enzyme 2 (ACE2) glycoprotein and facilitate host cell entry. We utilized glycomics-informed glycoproteomics to characterize site-specific microheterogeneity of glycosylation for a recombinant trimer Spike mimetic immunogen and for a soluble version of human ACE2. We combined this information with bioinformatics analyses of natural variants and with existing 3D structures of both glycoproteins to generate molecular dynamics simulations of each glycoprotein both alone and interacting with one another. Our results highlight roles for glycans in sterically masking polypeptide epitopes and directly modulating Spike-ACE2 interactions. Furthermore, our results illustrate the impact of viral evolution and divergence on Spike glycosylation, as well as the influence of natural variants on ACE2 receptor glycosylation. Taken together, these data can facilitate immunogen design to achieve antibody neutralization and inform therapeutic strategies to inhibit viral infection.

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Alpha-dystroglycan is a cell-surface glycoprotein that acts as a receptor for both extracellular matrix proteins containing laminin-G domains and certain arenaviruses. Receptor binding is thought to be mediated by a post-translational modification, and defective binding with laminin underlies a subclass of congenital muscular dystrophy. Here, using mass spectrometry-and NMR-based structural analyses, we identified a phosphorylated O-mannosyl glycan on the mucin-like domain of recombinant alpha-dystroglycan, which was required for laminin binding. We demonstrated that patients with muscle-eye-brain disease and Fukuyama congenital muscular dystrophy, as well as mice with myodystrophy, commonly have defects in a post-phosphoryl modification of this phosphorylated O-linked mannose, and that this modification is mediated by the likeacetylglucosaminyltransferase (LARGE) protein. Our findings expand our understanding of the mechanisms that underlie congenital muscular dystrophy.Diverse post-translational modifications influence the structure and function of many proteins. Dystroglycan (DG) is a membrane protein that requires extensive post-translational processing in order to function as an extracellular matrix receptor. It is comprised of an extracellular α-* To whom correspondence should be addressed. kevin-campbell@uiowa.edu.Supporting Online Material www.sciencemag.org Materials and Methods Figs. S1 to S12 Table S1 NIH Public Access DG subunit and a transmembrane β-DG subunit (1). α-DG serves as a receptor for extracellular matrix laminin G domain-containing ligands such as laminin (1) and agrin (2) in both muscle and brain, and these interactions depend on an unidentified post-translational α-DG modification. α-DG is also the cellular receptor for lymphocytic choriomeningitis virus (LCMV), Lassa fever virus (LFV), and clade C New World arenaviruses (3,4). Although the binding sites for LCMV and LFV on α-DG have not yet been identified, they are thought to overlap with the modification recognized by laminin (5,6).Glycosyltransferase-mediated glycosylation is one form of post-translational modification that can modulate protein structure and function. The main forms in mammals are N-and Oglycosylation, and these are distinguished by how the oligosaccharide moiety links to the amino acid. Mutations in six known or putative glycosyltransferase genes-POMT1 (7), POMT2 (8), POMGnT1 (9), fukutin (10), FKRP (11), and LARGE (12)-have been identified in patients with congenital muscular dystrophy (CMD). These disorders cover a spectrum of abnormalities affecting the brain, eye, and skeletal muscle, and show a dramatic gradient of phenotypic severity ranging from the most devastating in Walker-Warburg syndrome (WWS; OMIM# 236670), to less severe in muscle-eye-brain disease (MEB; OMIM# 253280) and Fukuyama CMD (FCMD; OMIM# 253800), and to mild limb-girdle muscular dystrophies. In these diseases, the ability of α-DG to bind laminin is markedly reduced (13), suggesting that these (putative) glycosyltransferases participa...

Identifying sites of post-translational modifications on proteins is a major challenge in proteomics. O-Linked ␤-N-acetylglucosamine (O-GlcNAc) is a dynamic nucleocytoplasmic modification more analogous to phosphorylation than to classical complex O-glycosylation. We describe a mass spectrometry-based method for the identification of sites modified by O-GlcNAc that relies on mild ␤-elimination followed by Michael addition with dithiothreitol (BEMAD). Using synthetic peptides, we also show that biotin pentylamine can replace dithiothreitol as the nucleophile. The modified peptides can be efficiently enriched by affinity chromatography, and the sites can be mapped using tandem mass spectrometry. This same methodology can be applied to mapping sites of serine and threonine phosphorylation, and we provide a strategy that uses modification-specific antibodies and enzymes to discriminate between the two post-translational modifications. The BEMAD methodology was validated by mapping three previously identified O-GlcNAc sites, as well as three novel sites, on Synapsin I purified from rat brain. BEMAD was then used on a purified nuclear pore complex preparation to map novel sites of O-GlcNAc modification on the Lamin B receptor and the nucleoporin Nup155. This method is amenable for performing quantitative mass spectrometry and can also be adapted to quantify cysteine residues. In addition, our studies emphasize the importance of distinguishing between O-phosphate versus O-GlcNAc when mapping sites of serine and threonine post-translational modification using ␤-elimination/Michael addition methods. The rapid identification of proteins by mass spectrometry has become commonplace in the postgenomic era (1). However, one major challenge that remains is the identification of post-translational modifications on these proteins. More than 25 years ago, Finn Wold and colleagues (2) recognized the abundance of naturally occurring modified forms of the genetically encoded 21 amino acids. In addition to phosphorylation, a variety of post-translational modifications, including acetylation (3), methylation (4), and O-linked ␤-N-acetylglucosamine (OGlcNAc) 1 (5-7), are now recognized to regulate protein functions in cellular processes. Therefore, identification of proteins along with their post-translational modifications, which has been referred to as "functional proteomics," is an important step in the characterization of proteomes. O-GlcNAc is a dynamic post-translational modification occurring on a variety of nucleocytoplasmic proteins and, in several instances, O-GlcNAc maps to the same or adjacent sites as phosphorylation (8, 9). Diverse classes of proteins are modified including cytoskeletal proteins, transcription factors, signaling adapter molecules, hormone receptors, nuclear pore complex (NPC) proteins, and kinases (10). The nucleocytoplasmic enzymes for the addition (O-GlcNAc transferase) and removal (neutral ␤-N-acetylglucosaminidase, O-GlcNAcase) of this modification have been cloned and characterized (11-16) and may ac...

SummaryArchaea, one of three major evolutionary lineages of life, encode proteasomes highly related to those of eukaryotes. In contrast, archaeal ubiquitin-like proteins are less conserved and not known to function in protein conjugation. This has complicated our understanding of the origins of ubiquitination and its connection to proteasomes. Here we report two small archaeal modifier proteins, SAMP1 and SAMP2, with a β-grasp fold and C-terminal diglycine motif similar to ubiquitin, that form protein-conjugates in the archaeon Haloferax volcanii. SAMP-conjugates were altered by nitrogen-limitation and proteasomal gene knockout and spanned various functions including components of the Urm1 pathway. LC-MS/MS-based collision-induced dissociation demonstrated isopeptide bonds between the C-terminal glycine of SAMP2 and the ε-amino group of lysines from a number of protein targets and Lys58 of SAMP2 itself, revealing poly-SAMP chains. The widespread distribution and diversity of pathways modified by SAMPylation suggest this type of protein-conjugation is central to the archaeal lineage.

The structural diversity of glycoprotein N-linked oligosaccharides is determined by the expression and regulation of glycosyltransferase activities and by the availability of the appropriate acceptor/donor substrates. Cells in different tissues and in different developmental stages utilize these control points to manifest unique glycan expression patterns in response to their surroundings. The activity of a Toll-like receptor, called Tollo/ Toll-8, induces a pattern of incompletely defined, but neural specific, glycan expression in the Drosophila embryo. Understanding the full extent of the changes in glycan expression that result from altered Tollo/Toll-8 signaling requires characterization of the complete N-linked glycan profile of both wild-type and mutant embryos. N-Linked glycans harvested from wildtype or mutant embryos were subjected to direct structural analysis by analytic and preparative high pressure liquid chromatography, by multidimensional mass spectrometry, and by exoglycosidase digestion, revealing a predominance of high mannose and paucimannose glycans. Di-, mono-, and nonfucosylated forms of hybrid, complex biantennary, and triantennary glycans account for 12% of the total wild-type glycan profile. Two sialylated glycans bearing N-acetylneuraminic acid were detected, the first direct demonstration of this modification in Drosophila. Glycan profiles change during normal development consistent with increasing ␣-mannosidase II and core fucosyltransferase enzyme activities, and with decreasing activity of the Fused lobes processing hexosaminidase. In tollo/toll-8 mutants, a dramatic, expected loss of difucosylated glycans is accompanied by unexpected decreases in monofucosylated and nonfucosylated hybrid glycans and increases in some nonfucosylated paucimannose and biantennary glycans. Therefore, tollo/toll-8 signaling influences flux through several processing steps that affect the maturation of N-linked glycans.Cell surface glycans mediate interactions between cells and define cellular identities within complex tissues at all stages of life (1-6). As embryonic cells differentiate and form organized tissues, glycan expression diversifies, generating glycosylation profiles that are specific for tissue and cell type (7-9). Mutations that affect oligosaccharide synthesis or processing result in neural deficits, skeletal/connective tissue abnormalities, anemia, compromised immune response, muscular dystrophy, or generalized failure to thrive (10 -14). The vital functions of cellular glycans and the pathophysiologic consequences of altered glycosylation emphasize the need for understanding the basic mechanisms that regulate glycan expression in intact organisms.The expanding characterization of glycosyltransferases in Drosophila melanogaster has begun to define the bounds of structural diversity in the glycan portfolio of the organism and has also generated new opportunities for genetically dissecting the mechanisms that control glycosylation. Loss-of-function mutations have been described in a handful...