Wolfgang Egge-Jacobsen

Broad spectrum O-linked protein glycosylation in the human pathogen Neisseria gonorrhoeae

Protein glycosylation is an important element of biologic systems because of its significant effects on protein properties and functions. Although prominent within all domains of life, O-linked glycosylation systems modifying serine and threonine residues within bacteria and eukaryotes differ substantially in target protein selectivity. In particular, well-characterized bacterial systems have been invariably dedicated to modification of individual proteins or related subsets thereof. Here we characterize a general O-linked glycosylation system that targets structurally and functionally diverse groups of membrane-associated proteins in the Gram-negative bacterium Neisseria gonorrhoeae, the etiologic agent of the human disease gonorrhea. The 11 glycoproteins identified here are implicated in activities as varied as protein folding, disulfide bond formation, and solute uptake, as well as both aerobic and anaerobic respiration. Along with their common trafficking within the periplasmic compartment, the protein substrates share quasi-related domains bearing signatures of low complexity that were demonstrated to encompass sites of glycan occupancy. Thus, as in eukaryotes, the broad scope of this system is dictated by the relaxed specificity of the glycan transferase as well as the bulk properties and context of the protein-targeting signal rather than by a strict amino acid consensus sequence. Together, these findings reveal previously unrecognized commonalities linking O-linked protein glycosylation in distantly related life forms.

Neisseria gonorrhoeae O-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure

Neisseria gonorrhoeae expresses an O‐linked protein glycosylation pathway that targets PilE, the major pilin subunit protein of the Type IV pilus colonization factor. Efforts to define glycan structure and thus the functions of pilin glycosylation (Pgl) components at the molecular level have been hindered by the lack of sensitive methodologies. Here, we utilized a ‘top‐down’ mass spectrometric approach to characterize glycan status using intact pilin protein from isogenic mutants. These structural data enabled us to directly infer the function of six components required for pilin glycosylation and to define the glycan repertoire of strain N400. Additionally, we found that the N. gonorrhoeae pilin glycan is O‐acetylated, and identified an enzyme essential for this unique modification. We also identified the N. gonorrhoeae pilin oligosaccharyltransferase using bioinformatics and confirmed its role in pilin glycosylation by directed mutagenesis. Finally, we examined the effects of expressing the PglA glycosyltransferase from the Campylobacter jejuni N‐linked glycosylation system that adds N‐acetylgalactosamine onto undecaprenylpyrophosphate‐linked bacillosamine. The results indicate that the C. jejuni and N. gonorrhoeae pathways can interact in the synthesis of O‐linked di‐ and trisaccharides, and therefore provide the first experimental evidence that biosynthesis of the N. gonorrhoeae pilin glycan involves a lipid‐linked oligosaccharide precursor. Together, these findings underpin more detailed studies of pilin glycosylation biology in both N. gonorrhoeae and N. meningitidis, and demonstrate how components of bacterial O‐ and N‐linked pathways can be combined in novel glycoengineering strategies.

Lysine methylation of VCP by a member of a novel human protein methyltransferase family.

Valosin-containing protein (VCP, also called p97) is an essential and highly conserved adenosine triphosphate-dependent chaperone implicated in a wide range of cellular processes in eukaryotes, and mild VCP mutations can cause severe neurodegenerative disease. Here we show that mammalian VCP is trimethylated on Lys315 in a variety of cell lines and tissues, and that the previously uncharacterized protein METTL21D (denoted here as VCP lysine methyltransferase, VCP-KMT) is the responsible enzyme. VCP methylation was abolished in three human VCP-KMT knockout cell lines generated with zinc-finger nucleases. Interestingly, VCP-KMT was recently reported to promote tumour metastasis, and indeed, VCP-KMT-deficient cells displayed reduced growth rate, migration and invasive potential. Finally, we present data indicating that VCP-KMT, calmodulin-lysine methyltransferase and eight uncharacterized proteins together constitute a novel human protein methyltransferase family. The present work provides new insights on protein methylation and its links to human disease.

Differential proteomic analysis of drought stress response in leaves of common bean (Phaseolus vulgaris L.)

The majority of common bean plants are cultivated under drought conditions. Maintaining crop yields under drought stress is thus one of the biggest challenges facing bean production. In order to improve our understanding of the complex mechanisms involved in the response of common bean (Phaseolus vulgaris) to drought stress, a proteomic approach was used to identify drought-responsive proteins in leaves of two cultivars differing in their response to drought, Tiber and more sensitive Starozagorski čern. 2D-DIGE was used to compare differences in protein abundance between control and stressed plants. Fifty-eight proteins whose abundance changed significantly were identified by LC-MS/MS in Tiber and 64 in Starozagorski čern. The majority of identified proteins were classified into functional categories that include energy metabolism, photosynthesis, ATP interconversion, protein synthesis and proteolysis, stress and defence related proteins. Details of the function of the identified proteins and their abundance profiles in Tiber and Starozagorski are discussed. Interactions between identified proteins were demonstrated by bioinformatics analysis, enabling a more complete insight into biological pathways and molecular functions affected by drought stress. The results form the basis for a further understanding of the biochemical mechanisms of drought response in common bean.

Efficacy and safety of short-term genistein intervention in patients with localized prostate cancer prior to radical prostatectomy: a randomized, placebo-controlled, double-blind Phase 2 clinical trial.

We conducted a placebo-controlled, block-randomized double-blind Phase 2 study to examine the effect of 30 mg synthetic genistein daily on serum and tissue biomarkers in patients with localized prostate cancer (CaP). Fifty-four study subjects were recruited and randomized to treatment with genistein (n = 23) or placebo (n = 24) for 3 to 6 wk prior to prostatectomy. Seven study subjects were noncompliant to the study protocol. Adverse events were few and mild. Serum prostate specific antigen (PSA) decreased by 7.8% in the genistein arm and increased by 4.4% in the placebo arm (P = 0.051). The PSA level was reduced in tumor tissue compared to normal tissue in the placebo arm. In the genistein arm, the PSA level in tumor and normal tissue was comparable. Total cholesterol was significantly lower in the genistein arm (P = 0.013). There were no significant effects on thyroid or sex hormones. Plasma concentrations of total genistein were on average 100-fold higher in the genistein arm after treatment (P < 0.001). Genistein at a dose that can be easily obtained from a diet rich in soy reduced the level of serum PSA in patients with localized CaP, without any effects on hormones. It was well tolerated and had a beneficial effect on blood cholesterol.

Neisseria gonorrhoeae Type IV pili undergo multisite, hierarchical modifications with phosphoethanolamine and phosphocholine requiring an enzyme structurally related to lipopolysaccharide phosphoethanolamine transferases

The zwitterionic phospho-forms phosphoethanolamine and phosphocholine are recognized as influential and important substituents of pathogen cell surfaces. PilE, the major pilin subunit protein of the type IV pilus (Tfp) colonization factor of Neisseria gonorrhoeae undergoes unique, post-translational modifications with these moieties. These phospho-form modifications have been shown to be O-linked alternately to a specific, conserved serine residue of PilE. However, the enzymes and precursors involved in their addition are unknown, and the full spectrum of PilE post-translational modifications has yet to be defined. Here, an intact protein-based mass spectrometric approach was integrated with bioinformatics and reverse genetics to address these matters. Specifically we show that a protein limited in its distribution to pathogenic Neisseria species and structurally related to enzymes implicated in phosphoethanolamine modification of lipopolysaccharide is necessary for PilE covalent modification with phosphoethanolamine and phosphocholine. These findings strongly suggest that protein phospho-form modification is mechanistically similar to processes underlying analogous modifications of prokaryotic saccharolipid glycans. We also show that PilE undergoes multisite and hierarchical phospho-form modifications and that the stoichiometries of site occupancy can be influenced by PilE primary structure and the abundance of the pilin-like protein PilV. Together, these findings have important implications for the structure and antigenicity of PilE.

Identification and Characterization of a Novel Human Methyltransferase Modulating Hsp70 Protein Function through Lysine Methylation

Background: The function of many proteins is regulated through post-translational methylation. Results: METTL21A was identified as a human protein methyltransferase targeting Hsp70 proteins, thereby altering their ability to interact with client proteins. Conclusion: METTL21A is a specific methyltransferase modulating the function of Hsp70 proteins. Significance: The activity of a human protein-modifying enzyme is unraveled, and the modification is demonstrated to have functional consequences. Hsp70 proteins constitute an evolutionarily conserved protein family of ATP-dependent molecular chaperones involved in a wide range of biological processes. Mammalian Hsp70 proteins are subject to various post-translational modifications, including methylation, but for most of these, a functional role has not been attributed. In this study, we identified the methyltransferase METTL21A as the enzyme responsible for trimethylation of a conserved lysine residue found in several human Hsp70 (HSPA) proteins. This enzyme, denoted by us as HSPA lysine (K) methyltransferase (HSPA-KMT), was found to catalyze trimethylation of various Hsp70 family members both in vitro and in vivo, and the reaction was stimulated by ATP. Furthermore, we show that HSPA-KMT exclusively methylates 70-kDa proteins in mammalian protein extracts, demonstrating that it is a highly specific enzyme. Finally, we show that trimethylation of HSPA8 (Hsc70) has functional consequences, as it alters the affinity of the chaperone for both the monomeric and fibrillar forms of the Parkinson disease-associated protein α-synuclein.

Poly(lactide-co-glycolide)-rifampicin nanoparticles efficiently clear Mycobacterium bovis BCG infection in macrophages and remain membrane-bound in phago-lysosomes.

Summary Nanoparticles (NPs) are increasingly used as biodegradable vehicles to selectively deliver therapeutic agents such as drugs or antigens to cells. The most widely used vehicle for this purpose is based on copolymers of lactic acid and glycolic acid (PLGA) and has been extensively used in experiments aimed at delivering antibiotics against Mycobacterium tuberculosis in animal models of tuberculosis. Here, we describe fabrication of PLGA NPs containing either a high concentration of rifampicin or detectable levels of the green fluorescent dye, coumarin-6. Our goal here was twofold: first to resolve the controversial issue of whether, after phagocytic uptake, PLGA NPs remain membrane-bound or whether they escape into the cytoplasm, as has been widely claimed. Second, we sought to make NPs that enclosed sufficient rifampicin to efficiently clear macrophages of infection with Mycobacterium bovis BCG. Using fluorescence microscopy and immuno-electron microscopy, in combination with markers for lysosomes, we show that BCG bacteria, as expected, localized to early phagosomes, but that at least 90% of PLGA particles were targeted to, and remained in, low pH, hydrolase-rich phago-lysosomes. Our data collectively argue that PLGA NPs remain membrane-enclosed in macrophages for at least 13 days and degrade slowly. Importantly, provided that the NPs are fabricated with sufficient antibiotic, one dose given after infection is sufficient to efficiently clear the BCG infection after 9–12 days of treatment, as shown by estimates of the number of bacterial colonies in vitro.

Genetic, structural, and antigenic analyses of glycan diversity in the O-linked protein glycosylation systems of human Neisseria species.

Bacterial capsular polysaccharides and lipopolysaccharides are well-established ligands of innate and adaptive immune effectors and often exhibit structural and antigenic variability. Although many surface-localized glycoproteins have been identified in bacterial pathogens and symbionts, it not clear if and how selection impacts associated glycoform structure. Here, a systematic approach was devised to correlate gene repertoire with protein-associated glycoform structure in Neisseria species important to human health and disease. By manipulating the protein glycosylation (pgl) gene content and assessing the glycan structure by mass spectrometry and reactivity with monoclonal antibodies, it was established that protein-associated glycans are antigenically variable and that at least nine distinct glycoforms can be expressed in vitro. These studies also revealed that in addition to Neisseria gonorrhoeae strain N400, one other gonococcal strain and isolates of Neisseria meningitidis and Neisseria lactamica exhibit broad-spectrum O-linked protein glycosylation. Although a strong correlation between pgl gene content, glycoform expression, and serological profile was observed, there were significant exceptions, particularly with regard to levels of microheterogeneity. This work provides a technological platform for molecular serotyping of neisserial protein glycans and for elucidating pgl gene evolution.

Automated, fast, and sensitive quantification of drugs in human plasma by LC/LC-MS: quantification of 6 protease inhibitors and 3 nonnucleoside transcriptase inhibitors.

An analytic assay based on automated sample preparation and liquid chromatography (LC) coupled with electrospray mass spectrometry (ESI-MS) was developed for the quantification of 6 protease inhibitors (PIs) and 3 nonnucleoside reverse transcriptase inhibitors (NNRTIs). The 6 PIs, amprenavir, indinavir, ritonavir, lopinavir, nelfinavir, and saquinavir, as well as the three NNRTIs, nevirapine, efavirenz, and delavirdine, require a succinct analysis technique for therapeutic drug monitoring in HIV/AIDS patients. After protein precipitation, samples were loaded on a C8, 10 × 4-mm extraction column, washed, and, after activation of the column-switching valve, backflushed onto the 30 × 2.1 mm C8 analytic column. [M+H]+ ions were detected in the selected ion mode. A nonlinear fit (y−1 = a + b/x, all r2 > 0.999) for amprenavir, indinavir, ritonavir, lopinavir, nelfinavir, and saquinavir and a linear fit (y = ax + b, all r2 > 0.999) for nevirapine, efavirenz, and delavirdine led to best regression. Absolute recoveries were as follows: PIs > 81%; NNRTIs > 76%. Interday and intraday precision were <12.5% for the PIs and <11.7% for the NNRTIs. Interday and intraday accuracy were <12.2% for the PIs and <14.9% for the NNRTIs. Limits of quantification were 20, 40, 50, 40, 40, 20, and 100 μg/L for amprenavir, indinavir, ritonavir, lopinavir, nelfinavir, saquinavir, and the NNRTIs, respectively. The assay allows fast analysis of patient samples for therapeutic drug monitoring (TDM) and has successfully been used for TDM and pharmacokinetic drug–drug interactions studies.