Rapidly evolving marmoset MSMB genes are differently expressed in the male genital tract

BACKGROUND: Beta-microseminoprotein, an abundant component in prostatic fluid, is encoded by the potential tumor suppressor gene MSMB. Some New World monkeys carry several copies of this gene, in contrast to most mammals, including humans, which have one only. Here we have investigated the background for the species difference by analyzing the chromosomal organization and expression of MSMB in the common marmoset (Callithrix jacchus). METHODS: Genes were identified in the Callithrix jacchus genome database using bioinformatics and transcripts were analyzed by RT-PCR and quantified by real time PCR in the presence of SYBR green. RESULTS: The common marmoset has five MSMB: one processed pseudogene and four functional genes. The latter encompass homologous genomic regions of 32-35 kb, containing the genes of 12-14 kb and conserved upstream and downstream regions of 14-19 kb and 3-4 kb. One gene, MSMB1, occupies the same position on the chromosome as the single human gene. On the same chromosome, but several Mb away, is another MSMB locus situated with MSMB2, MSMB3 and MSMB4 arranged in tandem. Measurements of transcripts demonstrated that all functional genes are expressed in the male genital tract, generating very high transcript levels in the prostate. The transcript levels in seminal vesicles and testis are two and four orders of magnitude lower. A single gene, MSMB3, accounts for more than 90% of MSMB transcripts in both the prostate and the seminal vesicles, whereas in the testis around half of the transcripts originate from MSMB2. These genes display rapid evolution with a skewed distribution of mutated nucleotides; in MSMB2 they affect nucleotides encoding the N-terminal Greek key domain, whereas in MSMB3 it is the C-terminal MSMB-unique domain that is affected. CONCLUSION: Callitrichide monkeys have four functional MSMB that are all expressed in the male genital tract, but the product from one gene, MSMB3, will predominate in seminal plasma. This gene and MSMB2, the predominating testicular gene, have accumulated mutations that affect different parts of the translation products, suggesting an ongoing molecular specialization that presumably yields functional differences in accessory sex glands and testis

β-Microseminoprotein Endows Post Coital Seminal Plasma with Potent Candidacidal Activity by a Calcium- and pH-Dependent Mechanism

The innate immune factors controlling Candida albicans are mostly unknown. Vulvovaginal candidiasis is common in women and affects approximately 70–75% of all women at least once. Despite the propensity of Candida to colonize the vagina, transmission of Candida albicans following sexual intercourse is very rare. This prompted us to investigate whether the post coital vaginal milieu contained factors active against C. albicans. By CFU assays, we found prominent candidacidal activity of post coital seminal plasma at both neutral and the acid vaginal pH. In contrast, normal seminal plasma did not display candidacidal activity prior to acidification. By antifungal gel overlay assay, one clearing zone corresponding to a protein band was found in both post coital and normal seminal plasma, which was subsequently identified as β-microseminoprotein. At neutral pH, the fungicidal activity of β-microseminoprotein and seminal plasma was inhibited by calcium. By NMR spectroscopy, amino acid residue E71 was shown to be critical for the calcium coordination. The acidic vaginal milieu unleashed the fungicidal activity by decreasing the inhibitory effect of calcium. The candidacidal activity of β-microseminoprotein was mapped to a fragment of the C-terminal domain with no structural similarity to other known proteins. A homologous fragment from porcine β-microseminoprotein demonstrated calcium-dependent fungicidal activity in a CFU assay, suggesting this may be a common feature for members of the β-microseminoprotein family. By electron microscopy, β-microseminoprotein was found to cause lysis of Candida. Liposome experiments demonstrated that β-microseminoprotein was active towards ergosterol-containing liposomes that mimic fungal membranes, offering an explanation for the selectivity against fungi. These data identify β-microseminoprotein as an important innate immune factor active against C. albicans and may help explain the low sexual transmission rate of Candida

The N-Terminal Amphipathic Helix of the Topological Specificity Factor MinE Is Associated with Shaping Membrane Curvature

Pole-to-pole oscillations of the Min proteins in Escherichia coli are required for the proper placement of the division septum. Direct interaction of MinE with the cell membrane is critical for the dynamic behavior of the Min system. In vitro, this MinE-membrane interaction led to membrane deformation; however, the underlying mechanism remained unclear. Here we report that MinE-induced membrane deformation involves the formation of an amphipathic helix of MinE2–9, which, together with the adjacent basic residues, function as membrane anchors. Biochemical evidence suggested that the membrane association induces formation of the helix, with the helical face, consisting of A2, L3, and F6, inserted into the membrane. Insertion of this helix into the cell membrane can influence local membrane curvature and lead to drastic changes in membrane topology. Accordingly, MinE showed characteristic features of protein-induced membrane tubulation and lipid clustering in in vitro reconstituted systems. In conclusion, MinE shares common protein signatures with a group of membrane trafficking proteins in eukaryotic cells. These MinE signatures appear to affect membrane curvature

Assessment of the higher order structure of Humira® Remicade® Avastin® Rituxan® Herceptin® and Enbrel® by 2D-NMR fingerprinting

The advent of monoclonal antibody biosimilar products has stimulated the development of analytical methods that can better characterize an important quality attribute, namely the higher order structure (HOS). Here, we propose a simple approach based on heteronuclear 2D NMR techniques at natural abundance for generating spectral fingerprints of the HOS at high resolution. We show that the proposed method can assess the HOS of six therapeutic products, adalimumab (Humira®), bevacizumab (Avastin®), infliximab (Remicade®), rituximab (Rituxan®), trastuzumab (Herceptin®), and Etanercept (Enbrel®). After treatment with immobilized papain, the purified fragments (Fab and Fc) were analyzed by 2D proton-nitrogen and proton-carbon NMR correlations. All Fab and Fc fragments produced high-resolution 2D-NMR spectra from which assessment of their higher order structure can be performed in the context of comparability studies. In particular, the two different sequences of Fc fragments could be unambiguously distinguished. The results show that it is possible to obtain structurally dependent information at amino acid resolution of these important therapeutic agents

A single N-acetylgalactosamine residue at threonine 106 modifies the dynamics and structure of interferon \u3b12a around the glycosylation site

Enzymatic addition of GalNAc to isotopically labeled IFN\u3b12a produced in Escherichia coli yielded the O-linked glycoprotein GalNAc\u3b1-[ 13C,15N]IFN\u3b12a. The three-dimensional structure of GalNAc\u3b1-IFN\u3b12a has been determined in solution by NMR spectroscopy at high resolution. Proton-nitrogen heteronuclear Overhauser enhancement measurements revealed that the addition of a single monosaccharide unit at Thr-106 significantly slowed motions of the glycosylation loop on the nanosecond time scale. Subsequent addition of a Gal unit produced Gal(\u3b21,3) GalNAc\u3b1[13C,15N]IFN\u3b12a. This extension resulted in a further decrease in the dynamics of this loop. The methodology used here allowed the first such description of the structure and dynamics of an O-glycoprotein and opens the way to the study of this class of proteins. \ua9 2013 by The American Society for Biochemistry and Molecular Biology, Inc.Peer reviewed: YesNRC publication: Ye

Appropriation of the MinD protein-interaction motif by the dimeric interface of the bacterial cell division regulator MinE

MinE is required for the dynamic oscillation of Min proteins that restricts formation of the cytokinetic septum to the midpoint of the cell in gram negative bacteria. Critical for this oscillation is MinD-binding by MinE to stimulate MinD ATP hydrolysis, a function that had been assigned to the first ∼30 residues in MinE. Previous models based on the structure of an autonomously folded dimeric C-terminal fragment suggested that the N-terminal domain is freely accessible for interactions with MinD. We report here the solution NMR structure of the full-length MinE dimer from Neisseria gonorrhoeae, with two parts of the N-terminal domain forming an integral part of the dimerization interface. Unexpectedly, solvent accessibility is highly restricted for residues that were previously hypothesized to directly interact with MinD. To delineate the true MinD-binding region, in vitro assays for MinE-stimulated MinD activity were performed. The relative MinD-binding affinities obtained for full-length and N-terminal peptides from MinE demonstrated that residues that are buried in the dimeric interface nonetheless participate in direct interactions with MinD. According to results from NMR spin relaxation experiments, access to these buried residues may be facilitated by the presence of conformational exchange. We suggest that this concealment of MinD-binding residues by the MinE dimeric interface provides a mechanism for prevention of nonspecific interactions, particularly with the lipid membrane, to allow the free diffusion of MinE that is critical for Min protein oscillation