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Poor lifestyle choices and genetic predisposition are factors that increase the number of cancer cases, one example being breast cancer, the third most diagnosed type of malignancy. Currently, there is a demand for the development of new strategies to ensure early detection and treatment options that could contribute to the complete remission of breast tumors, which could lead to increased overall survival rates. In this context, the glycans observed at the surface of cancer cells are presented as efficient tumor cell markers. These carbohydrate structures can be recognized by lectins which can act as decoders of the glycocode. The application of plant lectins as tools for diagnosis/treatment of breast cancer encompasses the detection and sorting of glycans found in healthy and malignant cells. Here, we present an overview of the most recent studies in this field, demonstrating the potential of lectins as: mapping agents to detect differentially expressed glycans in breast cancer, as histochemistry/cytochemistry analysis agents, in lectin arrays, immobilized in chromatographic matrices, in drug delivery, and as biosensing agents. In addition, we describe lectins that present antiproliferative effects by themselves and/or in conjunction with other drugs in a synergistic effect.

During mitosis, phosphorylation and dephosphorylation of lamins triggers the nuclear envelope disassembly/assembly. However, it hasn't been known whether lamin proteins undergo any modification other than phosphorylation during the cell cycle. Glycosylation of lamin proteins is one of the less studied post-translational modification. Glycosylation and phosphorylation compete for the same positions and interplay between two modifications generate a post-translational code in the cell. Based on this, we hypothesized that glycosylation of lamin A/C protein may be important in the regulation of the structural organization of the nuclear lamina during interphase and mitosis. We analysed the glycan units of lamin A/C protein in lung carcinoma cells synchronized at G2/M and S phases via CapLC-ESI-MS/MS. Besides, the outermost glycan units were determined using lectin blotting and gold-conjugated antibody and lectin staining. TEM studies also allowed us to observe the localization of glycosylated lamin A/C protein. With this study, we determined that lamin A/C protein shows O-glycosylation at G2/M and S phases of the cell cycle. In addition to O-GlcNAcylation and O-GalNAcylation, lamin A/C is found to be contain Gal, Fuc, Man, and Sia sugars at G2/M and S phases for the first time. Having found the glycan units of the lamin A/C protein suggests that glycosylation might have a role in the nuclear organization during the cell cycle., Ege University Scientific Research Projects Coordination [17-FEN-031], This work was supported by the Ege University Scientific Research Projects Coordination (grant number 17-FEN-031).

Background Von Willebrand factor (VWF) contains a number of free thiols, the majority of which are located in its C-domains, and these have been shown to alter VWF function, However, the impact of free thiols on function following acute exposure of VWF to collagen under high and pathological shear stress has not been determined. Methods VWF free thiols were blocked with N-ethylmaleimide and flow assays performed under high and pathological shear rates to determine the impact on platelet capture and collagen binding function. Atomic force microscopy (AFM) was used to probe the interaction of VWF with collagen and molecular simulations conducted to determine the effect of free thiols on the flexibility of the VWF-C4 domain. Results Blockade of VWF free thiols reduced VWF-mediated platelet capture to collagen in a shear-dependent manner, with platelet capture virtually abolished above 5000 s-1 and in regions of stenosis in microfluidic channels. Direct visualization of VWF fibers formed under extreme pathological shear rates and analysis of collagen-bound VWF attributed the effect to altered binding of VWF to collagen. AFM measurements showed that thiol-blockade reduced the lifetime and strength of the VWF-collagen bond. Pulling simulations of the VWF-C4 domain demonstrated that with one or two reduced disulphide bonds the C4 domain has increased flexibility and the propensity to undergo free-thiol exchange. Conclusions We conclude that free thiols in the C-domains of VWF enhance the flexibility of the molecule and enable it to withstand high shear forces following collagen binding, demonstrating a previously unrecognized role for VWF free thiols.

Data-independent acquisition (DIA) is now an emerging method in bottom–up proteomics and capable of achieving deep proteome coverage and accurate label-free quantification. However, for post-translational modifications, such as glycosylation, DIA methodology is still in the early stage of development. The full characterization of glycoproteins requires site-specific glycan identification as well as subsequent quantification of glycan structures at each site. The tremendous complexity of glycosylation represents a significant analytical challenge in glycoproteomics. This review focuses on the development and perspectives of DIA methodology for N- and O-linked glycoproteomics and posits that DIA-based glycoproteomics could be a method of choice to address some of the challenging aspects of glycoproteomics. First, the current challenges in glycoproteomics and the basic principles of DIA are briefly introduced. DIA-based glycoproteomics is then summarized and described into four aspects based on the actual samples. Finally, we discussed the important challenges and future perspectives in the field. We believe that DIA can significantly facilitate glycoproteomic studies and contribute to the development of future advanced tools and approaches in the field of glycoproteomics., Graphical Abstract, Highlights • Protein glycosylation and challenges in glycoproteomics. • Data-independent acquisition for deglycosylated and intact N-linked glycopeptides. • Unbiased screening of oxonium ions from all glycopeptide precursors. • Glyco–data-independent acquisition on mucin-type O-glycopeptides., In Brief As a highly abundant and diverse post-translational modification, protein glycosylation is challenging to characterize in various approaches including MS. In MS-based proteomics, data-independent acquisition (DIA) has been advanced rapidly and showed outstanding analytical performances. DIA now started to be applied in different facets of glycoproteomics, including deglycosylated and intact N-linked and O-linked glycopeptides, and screening of oxonium ions. We summarized current applications of DIA in glycoproteomics and discussed its limitations and perspectives.

Visual rhodopsins are membrane proteins that function as light photoreceptors in the vertebrate retina. Specific amino acids have been positively selected in visual pigments during mammal evolution, which, as products of adaptive selection, would be at the base of important functional innovations. We have analyzed the top candidates for positive selection at the specific amino acids and the corresponding reverse changes (F13M, Q225R and A346S) in order to unravel the structural and functional consequences of these important sites in rhodopsin evolution. We have constructed, expressed and immunopurified the corresponding mutated pigments and analyzed their molecular phenotypes. We find that position 13 is very important for the folding of the receptor and also for proper protein glycosylation. Position 225 appears to be important for the function of the protein affecting the G-protein activation process, and position 346 would also regulate functionality of the receptor by enhancing G-protein activation and presumably affecting protein phosphorylation by rhodopsin kinase. Our results represent a link between the evolutionary analysis, which pinpoints the specific amino acid positions in the adaptive process, and the structural and functional analysis, closer to the phenotype, making biochemical sense of specific selected genetic sequences in rhodopsin evolution., This work was supported by Ministerio de Ciencia e Innovación, Spain (grants SAF2011-30216-C02-01) and a grant from Fundación Ramón Areces (to PG), and Grups de Recerca Consolidats de la Generalitat de Catalunya (2009 SGR 1402 to PG), Ministerio de Economía y Competitividad, Spain (BFU2013-43726-P) and Departament d’Economia i Coneixement de la Generalitat de Catalunya (GRC 2014 SGR 866 to JB). MAFS is the recipient of an FPI-UPC predoctoral fellowship and BMI has been the recipient of a predoctoral fellowship from AGAUR, Generalitat de Catalunya (PhD grant 2011 FI BI 00275).

Olfactory receptors (ORs) constitute the largest family of sensory membrane proteins in mammals. They play a key role within the olfactory system in recognizing and discriminating a nearly unlimited number of structurally diverse odorous molecules. The molecular basis of OR-mediated signal detection and transduction is poorly understood. This is due to difficulties in functional expression of ORs in high yields, preventing structural and biophysical studies at the level of the receptor protein. Here we report on recombinant expression of mouse receptor mOR256-17 yielding 10(6) ORs per cell in transiently transfected mammalian cells. For quantification and optimization of OR expression, we employed different fluorescent probes. Green fluorescent protein fused to the C-terminus of mOR256-17 allowed quantification of total cellular OR biosynthesis, and post-translational fluorescence labeling of a 12-amino acid polypeptide sequence at the N-terminus permitted the selective visualization and quantification of ORs at the plasma membrane using cell flow cytometry. Our dual-color labeling approach is generally applicable to quantification of membrane proteins for mammalian cell-based expression. By screening a large odorant compound library, we discovered a selective spectrum of potent mOR256-17-specific agonists essential for probing the receptor function for future scaled-up productions.

Background Lentiviruses such as human and simian immunodeficiency viruses (HIV and SIV) undergo continual evolution in the host. Previous studies showed that the late-stage variants of SIV that evolve in one host replicate to significantly higher levels when transmitted to a new host. However, it is unknown whether HIVs or SIVs that have higher replication fitness are more genetically stable upon transmission to a new host. To begin to address this, we analyzed the envelope sequence variation of viruses that evolved in animals infected with variants of SIVMne that had been cloned from an index animal at different stages of infection. Results We found that there was more evolution of envelope sequences from animals infected with the late-stage, highly replicating variants than in animals infected with the early-stage, lower replicating variant, despite the fact that the late virus had already diversified considerably from the early virus in the first host, prior to transmission. Many of the changes led to the addition or shift in potential-glycosylation sites-, and surprisingly, these changes emerged in some cases prior to the detection of neutralizing antibody responses, suggesting that other selection mechanisms may be important in driving virus evolution. Interestingly, these changes occurred after the development of antibody whose anti-viral function is dependent on Fc-Fcγ receptor interactions. Conclusion SIV variants that had achieved high replication fitness and escape from neutralizing antibodies in one host continued to evolve upon transmission to a new host. Selection for viral variants with glycosylation and other envelope changes may have been driven by both neutralizing and Fcγ receptor-mediated antibody activities.