Use of spectral analysis to test hypotheses on the origin of pinnipeds

The evolutionary origin of the pinnipeds (seals, sea lions, and walruses) is still uncertain. Most authors support a hypothesis of a monophyletic origin of the pinnipeds from a caniform carnivore. A minority view suggests a diphyletic origin with true seals being related to the mustelids (otters and ferrets). The phylogenetic relationships of the walrus to other pinniped and carnivore families are also still particularly problematic. Here we examined the relative support for mono- and diphyletic hypotheses using DNA sequence data from the mitochondrial small subunit (12S) rRNA and cytochrome b genes. We first analyzed a small group of taxa representing the three pinniped families (Phocidae, Otariidae, and Odobenidae) and caniform carnivore families thought to be related to them. We inferred phylogenetic reconstructions from DNA sequence data using standard parsimony and neighbor-joining algorithms for phylogenetic inference as well as a new method called spectral analysis (Hendy and Penny) in which phylogenetic information is displayed independently of any selected tree. We identified and compensated for potential sources of error known to lead to selection of incorrect phylogenetic trees. These include sampling error, unequal evolutionary rates on lineages, unequal nucleotide composition among lineages, unequal rates of change at different sites, and inappropriate tree selection criteria. To correct for these errors, we performed additional transformations of the observed substitution patterns in the sequence data, applied more stringent structural constraints to the analyses, and included several additional taxa to help resolve long, unbranched lineages in the tree. We find that there is strong support for a monophyletic origin of the pinnipeds from within the caniform carnivores, close to the bear/raccoon/panda radiation. Evidence for a diphyletic origin was very weak and can be partially attributed to unequal nucleotide compositions among the taxa analyzed. Subsequently, there is slightly more evidence for grouping the walrus with the eared seals versus the true seals. A more conservative interpretation, however, is that the walrus is an early, but not the first, independent divergence from the common pinniped ancestor.      

NMR characterization of conformational fluctuations and noncovalent interactions of SUMO protein from Drosophila melanogaster (dSmt3)

Structural heterogeneity in the native-state ensemble of dSmt3, the only small ubiquitin-like modifier (SUMO) in Drosophila melanogaster, was investigated and compared with its human homologue SUMO1. Temperature dependence of amide proton's chemical shift was studied to identify amino acids possessing alternative structural conformations in the native state. Effect of small concentration of denaturant (1M urea) on this population was also monitored to assess the ruggedness of near-native energy landscape. Owing to presence of many such amino acids, especially in the β₂-loop-α region, the native state of dSmt3 seems more flexible in comparison to SUMO1. Information about backbone dynamics in ns-ps timescale was quantified from the measurement of ¹⁵N-relaxation experiments. Furthermore, the noncovalent interaction of dSmt3 and SUMO1 with Daxx12 (Daxx⁷²⁹DPEEIIVLSDSD⁷⁴⁰), a [V/I]-X-[V/I]-[V/I]-based SUMO interaction motif, was characterized using Bio-layer Interferometery and NMR spectroscopy. Daxx12 fits itself in the groove formed by β₂-loop-α structural region in both dSmt3 and SUMO1, but the binding is stronger with the former. Flexibility of β₂-loop-α region in dSmt3 is suspected to assist its interaction with Daxx12. Our results highlight the role of native-state flexibility in assisting noncovalent interactions of SUMO proteins especially in organisms where a single SUMO isoform has to tackle multiple substrates single handedly.     

Glycosylation of zona pellucida glycoprotein-3 is required for inducing acrosomal exocytosis in the bonnet monkey.

To investigate the role of polypeptide backbone vis-à-vis glycosylation of the putative primary sperm receptor, the bonnet monkey (Macaca radiata) zona pellucida glycoprotein-3 (bmZP3), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was expressed as polyhistidine fusion protein either in Escherichia coli orusing baculovirus expression system. The recombinant bmZP3 (r-bmZP3) was purified using nickel-nitrilotriacetic acid resin and subsequently refolded in the presence of oxidized and reduced glutathione. SDS-PAGE and Western blot analysis revealed approximately 43 and approximately 52 kDa bands corresponding to E. coli and baculovirus expressed r-bmZP3, respectively. The r-bmZP3 purified from both E. coli and baculovirus binds to the principal segment of the acrosomal cap of the capacitated bonnet monkey spermatozoa as evaluated by indirect immunofluoresence assay. In a competitive inhibition assay, the binding of biotinylated baculovirus expressed r-bmZP3 to capacitated spermatozoa was inhibited not only by cold baculovirus expressed r-bmZP3 but also by E. coli expressed r-bmZP3 and vice-versa. Lectin binding studies revealed that the baculovirus r-bmZP3 has N-linked glycosylation with galactose and mannose residues. Capacitated spermatozoa, in the presence of baculovirus expressed r-bZP3, undergoes a significant increase (p < 0.01) in the acrosomal exocytosis as compared to control whereas E. coli expressed r-bmZP3 failed to have a significant effect. These results suggest that though the polypeptide backbone of ZP3 is sufficient for its binding to capacitated spermatozoa, yet glycosylation is required for induction of acrosomal exocytosis.     

Efficacy of antibodies against a chimeric synthetic peptide encompassing epitopes of bonnet monkey (Macaca radiata) zona pellucida-1 and zona pellucida-3 glycoproteins to inhibit in vitro human sperm-egg binding

Immunocontraception achieved by immunization with zona pellucida (ZP) glycoproteins is invariably associated with ovarian dysfunction. Use of ZP glycoprotein-based synthetic peptides as immunogens has been proposed to overcome adverse side effects on ovaries. In the present study, a chimeric peptide encompassing the epitopes of bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1; amino acid residues 251-273) and ZP glycoprotein-3 (bmZP3; amino acid residues 324-347), separated by a tri-glycine spacer, was synthesized and conjugated to diphtheria toxoid (DT). Immunization of female BALB/cJ mice and bonnet monkeys with the chimeric peptide led to generation of antibodies that reacted with the chimeric peptide, individual bmZP1 & bmZP3 peptides, and also recombinant bmZP1 and bmZP3 proteins expressed by E. coli in an ELISA. Indirect immunofluorescence studies revealed that the immune serum also recognized human as well as bonnet monkey ZP. A significant inhibition of human sperm binding to ZP was observed with antibodies generated against the chimeric peptide in mice (P = 0.0001) as well as monkeys (P = 0.0002) in a hemizona assay (HZA). The inhibition efficacy was significantly higher than that observed by using antibodies against the individual bmZP1 and bmZP3 peptides. Interestingly, no ovarian pathology was observed in female bonnet monkeys immunized with the chimeric peptide. These studies have demonstrated that the chimeric peptide encompassing peptides of multiple ZP glycoproteins may be a promising candidate antigen for designing immunocontraceptive vaccines.     

The Lectin Domain of the Polypeptide GalNAc Transferase Family of Glycosyltransferases (ppGalNAc Ts) Acts as a Switch Directing Glycopeptide Substrate Glycosylation in an N- or C-terminal Direction,Further Controlling Mucin Type O-Glycosylation

Mucin type O-glycosylation is initiated by a large family of polypeptide GalNAc transferases (ppGalNAc Ts) that add α-GalNAc to the Ser and Thr residues of peptides. Of the 20 human isoforms, all but one are composed of two globular domains linked by a short flexible linker: a catalytic domain and a ricin-like lectin carbohydrate binding domain. Presently, the roles of the catalytic and lectin domains in peptide and glycopeptide recognition and specificity remain unclear. To systematically study the role of the lectin domain in ppGalNAc T glycopeptide substrate utilization, we have developed a series of novel random glycopeptide substrates containing a single GalNAc-O-Thr residue placed near either the N or C terminus of the glycopeptide substrate. Our results reveal that the presence and N- or C-terminal placement of the GalNAc-O-Thr can be important determinants of overall catalytic activity and specificity that differ between transferase isoforms. For example, ppGalNAc T1, T2, and T14 prefer C-terminally placed GalNAc-O-Thr, whereas ppGalNAc T3 and T6 prefer N-terminally placed GalNAc-O-Thr. Several transferase isoforms, ppGalNAc T5, T13, and T16, display equally enhanced N- or C-terminal activities relative to the nonglycosylated control peptides. This N- and/or C-terminal selectivity is presumably due to weak glycopeptide binding to the lectin domain, whose orientation relative to the catalytic domain is dynamic and isoform-dependent. Such N- or C-terminal glycopeptide selectivity provides an additional level of control or fidelity for the O-glycosylation of biologically significant sites and suggests that O-glycosylation may in some instances be exquisitely controlled.     

Acceptor specificities and selective inhibition of recombinant human Gal- and GlcNAc-transferases that synthesize core structures 1, 2, 3 and 4 of O-glycans

Background

Modifications of proteins by O-glycosylation determine many of the properties and functions of proteins. We wish to understand the mechanisms of O-glycosylation and develop inhibitors that could affect glycoprotein functions and alter cellular behavior.

Methods

We expressed recombinant soluble human Gal- and GlcNAc-transferases that synthesize the O-glycan cores 1 to 4 and are critical for the overall structures of O-glycans. We determined the properties and substrate specificities of these enzymes using synthetic acceptor substrate analogs. Compounds that were inactive as substrates were tested as inhibitors.

Results

Enzymes significantly differed in their recognition of the sugar moieties and aglycone groups of substrates. Core 1 synthase was active with glycopeptide substrates but GlcNAc-transferases preferred substrates with hydrophobic aglycone groups. Chemical modifications of the acceptors shed light on enzyme–substrate interactions. Core 1 synthase was weakly inhibited by its substrate analog benzyl 2-butanamido-2-deoxy-α-d-galactoside while two of the three GlcNAc-transferases were selectively and potently inhibited by bis-imidazolium salts which are not substrate analogs.

Conclusions

This work delineates the distinct specificities and properties of the enzymes that synthesize the common O-glycan core structures 1 to 4. New inhibitors were found that could selectively inhibit the synthesis of cores 1, 2 and 3 but not core 4.

General significance

These studies help our understanding of the mechanisms of action of enzymes critical for O-glycosylation. The results may be useful for the re-engineering of O-glycosylation to determine the roles of O-glycans and the enzymes critical for O-glycosylation, and for biotechnology with potential therapeutic applications.     

The impact of operative approach on outcome of surgery for gastro-oesophageal tumours

Background

Caveolin-1 is thought to have an important impact on both signal transduction and mediation of intracellular processes. Furthermore, it has been suggested that Caveolin-1 may contribute to certain steps of carcinogenesis in various types of cancer. We examined the potential clinical relevance of Caveolin-1 in normal, benign and malignant breast tissue specimens.

Methods

Using tissue microarray (TMA) technology cases of invasive breast cancer, DCIS, benign breast disease (i.e. fibroadenoma, sclerosing adenosis, ductal hyperplasia and radial scar) and normal breast tissue were evaluated for Caveolin-1 expression. Immunohistochemical staining with an anti-Caveolin-1-antibody was performed. Staining intensity was quantified semiquantitatively. In invasive lesions staining results were correlated with clinical and pathological data.

Results

No Caveolin-1 expression was observed in epithelial cells of normal breast tissue (n = 5), benign breast disease (n = 295) and DCIS (n = 108). However, Caveolin-1 expression was found in 32 of 109 cases of invasive breast carcinomas (29.4%). Caveolin-1 expression in invasive breast cancer could neither be correlated with survival parameters such as overall or disease-free survival nor with established clinical and pathological markers.

Conclusion

In this study we demonstrated expression of Caveolin-1 in one third of invasive breast cancers. A significant increase in Caveolin-1 expression was observed comparing invasive breast cancer to both benign breast tissue and non-invasive breast cancer. Since inhibitors of Caveolin-1 signalling are available, targeting Caveolin-1 in breast cancer may represent a potential option for future breast cancer treatment.