Targeted identification of glycosylated proteins in the gastric pathogen helicobacter pylori (Hp)

Virulence of the gastric pathogen Helicobacter pylori (Hp) is directly linked to the pathogen\u27s ability to glycosylate proteins; for example, Hp flagellin proteins are heavily glycosylated with the unusual nine-carbon sugar pseudaminic acid, and this modification is absolutely essential for Hp to synthesize functional flagella and colonize the host\u27s stomach. Although Hp\u27s glycans are linked to pathogenesis, Hp\u27s glycome remains poorly understood; only the two flagellin glycoproteins have been firmly characterized in Hp. Evidence from our laboratory suggests that Hp synthesizes a large number of as-yet unidentified glycoproteins. Here we set out to discover Hp\u27s glycoproteins by coupling glycan metabolic labeling with mass spectrometry analysis. An assessment of the subcellular distribution of azide-labeled proteins by Western blot analysis indicated that glycoproteins are present throughout Hp and may therefore serve diverse functions. To identify these species, Hp\u27s azide-labeled glycoproteins were tagged via Staudinger ligation, enriched by tandem affinity chromatography, and analyzed by multidimensional protein identification technology. Direct comparison of enriched azide-labeled glycoproteins with a mock-enriched control by both SDS-PAGE and mass spectrometry-based analyses confirmed the selective enrichment of azide-labeled glycoproteins. We identified 125 candidate glycoproteins with diverse biological functions, including those linked with pathogenesis. Mass spectrometry analyses of enriched azide-labeled glycoproteins before and after cleavage of O-linked glycans revealed the presence of Staudinger ligation-glycan adducts in samples only after beta-elimination, confirming the synthesis of O-linked glycoproteins in Hp. Finally, the secreted colonization factors urease alpha and urease beta were biochemically validated as glycosylated proteins via Western blot analysis as well as by mass spectrometry analysis of cleaved glycan products. These data set the stage for the development of glycosylation-based therapeutic strategies, such as new vaccines based on natively glycosylated Hp proteins, to eradicate Hp infection. Broadly, this report validates metabolic labeling as an effective and efficient approach for the identification of bacterial glycoproteins. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc

Mechanism and timing of Mcm2–7 ring closure during DNA replication origin licensing

The opening and closing of two ring-shaped Mcm2-7 DNA helicases is necessary to license eukaryotic origins of replication, although the mechanisms controlling these events are unclear. The origin-recognition complex (ORC), Cdc6 and Cdt1 facilitate this process by establishing a topological link between each Mcm2-7 hexamer and origin DNA. Using colocalization single-molecule spectroscopy and single-molecule Förster resonance energy transfer (FRET), we monitored ring opening and closing of Saccharomyces cerevisiae Mcm2-7 during origin licensing. The two Mcm2-7 rings were open during initial DNA association and closed sequentially, concomitant with the release of their associated Cdt1. We observed that ATP hydrolysis by Mcm2-7 was coupled to ring closure and Cdt1 release, and failure to load the first Mcm2-7 prevented recruitment of the second Mcm2-7. Our findings identify key mechanisms controlling the Mcm2-7 DNA-entry gate during origin licensing, and reveal that the two Mcm2-7 complexes are loaded via a coordinated series of events with implications for bidirectional replication initiation and quality control.National Institutes of Health (U.S.) (Grant R01 GM52339)National Institutes of Health (U.S.) (Pre-Doctoral Training Grant GM007287)National Cancer Institute (U.S.) (Koch Institute Support Grant P30-CA14051

Mechanism and importance of Mcm2-7 double-hexamer formation during DNA replication initiation

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2019Cataloged from PDF version of thesis.Includes bibliographical references.All cells must duplicate their genome completely and accurately in each cell cycle. Thus, DNA replication is a highly-regulated multi-step process that ensures the genome is duplicated only once per cell cycle. In eukaryotic cells, initiation of DNA replication begins with loading of two heterohexameric Mcm2-7 helicases around origin DNA during G1 phase. The two helicases are loaded in opposite orientations and interact with each other at their N-terminal domains to form a head-to-head "double hexamer". In S phase, the helicases are activated by helicase-activation proteins to initiate DNA unwinding. Importantly, this event is the committed step of replication initiation. Loading of two helicases in the head-to-head double hexamer ensures DNA unwinding on both sides of the origin and allows the assembly of bi-directional forks essential for complete DNA replication. Two Mcm2-7 helicases are loaded onto the DNA sequentially.The order of events during the first helicase loading has been established, but the mechanism of double-hexamer formation remains unclear. Because the two helicases interact at their N-terminal domains, these regions represent potential mediators of double-hexamer formation. This thesis outlines the potential mechanism and the importance of double-hexamer formation. A conserved motif within Mcm2-7 N-terminal region is required for stable double-hexamer formation and cell viability. Single-molecule analyses of Mcm2-7 containing a mutation within this motif indicated that this mutant form double-hexamer interactions briefly before the two hexamers come apart. Interestingly, after double-hexamer dissolution, the two mutant helicases do not form subsequent double-hexamer interaction. Both wild-type and the mutant Mcm2-7 exhibit double-hexamer interaction rapidly after the arrival of the second Mcm2-7.Together, these data support the model that double-hexamer formation is coordinated with loading of the second Mcm2-7. Finally, the requirement of the double hexamer during helicase activation was investigated using Mcm2-7 complex containing the mutant that inhibits double-hexamer formation. The double hexamer is not essential for recruitment of three critical helicase-activation proteins, but it is required for initial origin DNA unwinding. These findings identify a crucial motif for stable double-hexamer formation and suggest that DNA unwinding is the first step in replication initiation that requires double-hexamer form of the helicases.by Kanokwan Champasa.Ph. D.Ph.D. Massachusetts Institute of Technology, Department of Biolog

A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding

Licensing of eukaryotic origins of replication requires DNA loading of two copies of the McM2-7 replicative helicase to form a head-to-head double-hexamer, ensuring activated helicases depart the origin bidirectionally. To understand the formation and importance of this double-hexamer, we identified mutations in a conserved and essential McM4 motif that permit loading of two McM2-7 complexes but are defective for double-hexamer formation. Single-molecule studies show mutant McM2-7 forms initial hexamer-hexamer interactions; however, the resulting complex is unstable. Kinetic analyses of wild-type and mutant McM2-7 reveal a limited time window for double-hexamer formation following second McM2-7 association, suggesting that this process is facilitated. Double-hexamer formation is required for extensive origin DNA unwinding but not initial DNA melting or recruitment of helicase-activation proteins (Cdc45, GINS, McM10). Our findings elucidate dynamic mechanisms of origin licensing, and identify the transition between initial DNA melting and extensive unwinding as the first initiation event requiring double-hexamer formation.National Institute of General Medical Sciences (Grant GM52339)National Cancer Institute (Grant P30-CA14051