Identification of N -Linked Glycosylation Sites in Human Testis Angiotensin-converting Enzyme and Expression of an Active Deglycosylated Form

The sites of glycosylation of Chinese hamster ovary cell expressed testicular angiotensin-converting enzyme (tACE) have been determined by matrix-assisted laser desorption ionization/time of flight/mass spectrometry of peptides generated by proteolytic and cyanogen bromide digestion. Two of the seven potential N-linked glycosylation sites, Asn90 and Asn109, were found to be fully glycosylated by analysis of peptides before and after treatment with a series of glycosidases and with endoproteinase Asp-N. The mass spectra of the glycopeptides exhibit characteristic clusters of peaks which indicate the N-linked glycans in tACE to be mostly of the biantennary, fucosylated complex type. This structural information was used to demonstrate that three other sites, Asn155, Asn337, and Asn586, are partially glycosylated, whereas Asn72 appears to be fully glycosylated. The only potential site that was not modified is Asn620. Sequence analysis of tryptic peptides obtained from somatic ACE (human kidney) identified six glycosylated and one unglycosylated Asn. Only one of these glycosylation sites had a counterpart in tACE. Comparison of the two proteins reveals a pattern in which amino-terminal N-linked sites are preferred. The functional significance of glycosylation was examined with a tACE mutant lacking the O-glycan-rich first amino-terminal 36 residues and truncated at Ser625. When expressed in the presence of the alpha-glucosidase I inhibitor N-butyldeoxynojirimycin and treated with endoglycosidase H to remove all but the terminal N-acetylglucosamine residues, it retained full enzymatic activity, was electrophoretically homogeneous, and is a good candidate for crystallographic studies

UniDecCD: Deconvolution of Charge Detection-Mass Spectrometry Data

Native mass spectrometry (MS) has become a versatile tool for characterizing high-mass complexes and measuring biomolecular interactions. Native MS usually requires resolution of different charge states produced by electrospray ionization to measure the mass, which is difficult for highly heterogeneous samples that have overlapping and unresolvable charge states. Charge detection-mass spectrometry (CD-MS) seeks to address this challenge by simultaneously measuring the charge and m/z for isolated ions. However, CD-MS often shows uncertainty in the charge measurement that limits the resolution. To overcome this charge state uncertainty, we developed UniDecCD (UCD) software for computational de-convolution of CD-MS data, which significantly improves the resolution of CD-MS data. Here, we describe the UCD algorithm and demonstrate its ability to improve CD-MS resolution of proteins, megadalton viral capsids, and heterogeneous nanodiscs made from natural lipid extracts. UCD provides a user-friendly interface that will increase the accessibility of CD-MS technology and provide a valuable new computational tool for CD-MS data analysis

Roles of a novel Crp/Fnr family transcription factor Lmo0753 in soil survival, biofilm production and surface attachment to fresh produce of Listeria monocytogenes.

Listeria monocytogenes is a foodborne bacterial pathogen and the causative agent of an infectious disease, listeriosis. L. monocytogenes is ubiquitous in nature and has the ability to persist in food processing environments for extended periods of time by forming biofilms and resisting industrial sanitization. Human listeriosis outbreaks are commonly linked to contaminated dairy products, ready-to-eat meats, and in recent years, fresh produce such as lettuce and cantaloupes. We identified a putative Crp/Fnr family transcription factor Lmo0753 that is highly specific to human-associated genetic lineages of L. monocytogenes. Lmo0753 possesses two conserved functional domains similar to the major virulence regulator PrfA in L. monocytogenes. To determine if Lmo0753 is involved in environmental persistence-related mechanisms, we compared lmo0753 deletion mutants with respective wild type and complementation mutants of two fully sequenced L. monocytogenes genetic lineage II strains 10403S and EGDe for the relative ability of growth under different nutrient availability and temperatures, soil survival, biofilm productivity and attachment to select fresh produce surfaces including romaine lettuce leaves and cantaloupe rinds. Our results collectively suggested that Lmo0753 plays an important role in L. monocytogenes biofilm production and attachment to fresh produce, which may contribute to the environmental persistence and recent emergence of this pathogen in human listeriosis outbreaks linked to fresh produce

Bacterial strains used in this study.

<p>Bacterial strains used in this study.</p

<i>L. monocytogenes</i> soil survival.

<p>Bacterial survival in potting soil at 25°C for 30 days. Standard deviations represent three independent experiments performed in triplicate.</p

<i>L. monocytogenes</i> attachment to fresh produce surface.

<p>A) Bacterial attachment to romaine lettuce. B) Bacterial attachment to cantaloupe rind. Standard deviations represent three independent experiments. Significant differences in comparison to the parent strains under the same condition are shown as * (<i>P</i><0.5), ** (<i>P</i><0.001), and *** (<i>P</i><0.0001).</p

Growth of <i>L. monocytogenes</i> strains in broth culture.

<p>Standard deviations represent three independent experiments.</p