SMAR1 favors immunosurveillance of cancer cells by modulating calnexin and MHC I expression.

Down-regulation or loss of MHC class I expression is a major mechanism used by cancer cells to evade immunosurveillance and increase their oncogenic potential. MHC I mediated antigen presentation is a complex regulatory process, controlled by antigen processing machinery (APM) dictating immune response. Transcriptional regulation of the APM that can modulate gene expression profile and their correlation to MHC I mediated antigen presentation in cancer cells remain enigmatic. Here, we reveal that Scaffold/Matrix-Associated Region 1- binding protein (SMAR1), positively regulates MHC I surface expression by down-regulating calnexin, an important component of antigen processing machinery (APM) in cancer cells. SMAR1, a bonafide MAR binding protein acts as a transcriptional repressor of several oncogenes. It is down-regulated in higher grades of cancers either through proteasomal degradation or through loss of heterozygosity (LOH) at the Chr.16q24.3 locus where the human homolog of SMAR1 (BANP) has been mapped. It binds to a short MAR region of the calnexin promoter forming a repressor complex in association with GATA2 and HDAC1. A reverse correlation between SMAR1 and calnexin was thus observed in SMAR1-LOH cells and also in tissues from breast cancer patients. To further extrapolate our findings, influenza A (H1N1) virus infection assay was performed. Upon viral infection, the levels of SMAR1 significantly increased resulting in reduced calnexin expression and increased MHC I presentation. Taken together, our observations establish that increased expression of SMAR1 in cancers can positively regulate MHC I surface expression thereby leading to higher chances of tumor regression and elimination of cancer cells.

Scorpion Venom-Toxins that Aid in Drug Development: A Review.

Scorpion venom components have multifaceted orientation against bacterial, viral, fungal infections and other neuronal disorders. They can modulate the ion channels (K+, Na+, Cl-, Ca2+) of our body and this concept has been hypothesized in formulating pharmaceuticals. The triumphant achievement of these venom components as formulated anticancer agent in Phase I and Phase II clinical trials allure researchers to excavate beneficial venom components prohibiting DNA replication in malignant tumor cells. This review brings forth the achievements of Science and Technology in classifying the venom components as therapeutics and further application in drug product development.

Absolute Quantitation of Glycoforms of Two Human IgG Subclasses Using Synthetic Fc Peptides and Glycopeptides.

Immunoglobulins, such as immunoglobulin G (IgG), are of prime importance in the immune system. Polyclonal human IgG comprises four subclasses, of which IgG1 and IgG2 are the most abundant in healthy individuals. In an effort to develop an absolute MALDI-ToF-MS quantitative method for these subclasses and their Fc N-glycoforms, (glyco)peptides were synthesized using a solid-phase approach and used as internal standards. Tryptic digest glycopeptides from monoclonal IgG1 and IgG2 samples were first quantified using EEQYN(GlcNAc)STYR and EEQFN(GlcNAc)STFR standards, respectively. For IgG1, a similar glycopeptide where tyrosine (Y) was isotopically labelled was used to quantify monoclonal IgG1 that had been treated with the enzyme Endo-F2, i.e., yielding tryptic glycopeptide EEQYN(GlcNAc)STYR. The next step was to quantify single subclasses within polyclonal human IgG samples. Although ion abundances in the MALDI spectra often showed higher signals for IgG2 than IgG1, depending on the spotting solvent used, determination of amounts using the newly developed quantitative method allowed to obtain accurate concentrations where IgG1 species were predominant. It was observed that simultaneous analysis of IgG1 and IgG2 yielded non-quantitative results and that more success was obtained when subclasses were quantified one by one. More experiments served to assess the respective extraction and ionization efficiencies of EEQYNSTYR/EEQFNSTFR and EEQYN(GlcNAc)STYR/EEQFN(GlcNAc)STFR mixtures under different solvent and concentration conditions. Graphical Abstract ᅟ.

Multiplexed azido-group isotopic capture (MAGIC) beads: Selective analysis of azido compounds using a propargyl-based cleavable linker, a proof of concept.

RATIONALE: A cleavable linker is designed and synthesized for the selective capture of azide-containing compounds. This article presents a proof of concept methodology involving the use of peptide-functionalized aminopropyl silica, on which the peptide is constructed by solid-phase peptide synthesis. METHODS: The peptide linker has L-propargylglycine (Pra) at one terminal end to allow the conjugation of azide-containing molecules by copper assisted azide alkyne cycloaddition, also known as click reaction. L-Arginine (Arg) is placed just before Pra to permit the release of the captured product by tryptic cleavage. Three glycine (Gly) residues, as part of the linker, are appended to the silica bead to present a spacer section that allows efficient tryptic cleavage devoid of steric hindrance imposed by the bulky bead. The bead composition is Si-O-propyl-NH-Gly-Gly-Gly-Arg-Pra. RESULTS: This solid-phase material can be used to capture and release azide-functionalized compounds. The beads are first tested on three azido compounds, 2-azido-2-deoxyglucose (ADG), BOC-p-azido-Phe-OH (BAzPhe), where BOC = tert-butoxycarbonyl, and tetraacetylated-N-azidomannosamine (Ac4 ManNAz). Copper-mediated click reaction conditions are used and released products are characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem MS (MS/MS). CONCLUSIONS: This method allows easy identification of captured compounds based on mass and fragmentation analysis. Moreover, it is useful for the analysis of small azide-containing compounds by MALDI-TOF-MS which may not be possible otherwise due to matrix interferences. The insertion of isotopically labeled Arg residues provides the possibility of multiplex analysis, from which the beads have been called MAGIC (for Multiplexed Azido-Group Isotopic Capture). Copyright © 2016 John Wiley & Sons, Ltd.

Characterization of immunoglobulins through analysis of N-glycopeptides by MALDI-TOF MS.

The aim of this report is to emphasize the role, usefulness and power of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in the analysis of glycoforms of antibodies (Abs) through their proteolytic glycopeptides. Abs are complex biomolecules in which glycans hold determinant properties and thus need to be thoroughly characterized following Ab production by recombinant methods or Ab collection from human/animal serum or tissue. In spite of the great robustness of MALDI-TOF MS in terms of tolerance to impurities, the analysis of Abs and Ab components using this technique requires extensive sample preparation involving all or some of chromatography, solid phase extraction, enzymatic modification, and chemical derivatization. This report focuses on a monoclonal Ab produced in cell culture, as well as on a polyclonal human immunoglobulin (Ig) G obtained commercially and a polyclonal porcine IgG obtained from serum. A method is first provided to separate Ab protein chain components (light chains, heavy chains) by gel electrophoresis, which is useful for instance for protein-A eluates of Igs either from cell culture or biological samples. This allows for in-gel proteolytic digestion of the protein gel band(s) of choice for further MS characterization. Also discussed is the more conventional in-solution overnight digestion method used here with each of two proteolytic enzymes, i.e. trypsin and chymotrypsin. The overnight method is in turn compared with a much faster approach, that of digesting Abs with trypsin or chymotrypsin through the action of microwave heating. For method comparison, glycopeptides are fractionated from digestion mixtures using mostly C-18 cartridges for simplicity, although this enrichment procedure is also compared with other published procedures. The advantages of MALDI tandem mass spectrometry are highlighted for glycopeptide analysis, and lastly an esterification method applied to glycopeptides is discussed for retention of sialic acid residues on peptide acidic glycoforms.

A systematic study of glycopeptide esterification for the semi-quantitative determination of sialylation in antibodies.

RATIONALE: In the expression of recombinant proteins, an important parameter to control or influence is their level of sialylation. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric (MS) methods tend to either underestimate (positive mode) or overestimate (negative mode) the content of sialylated vs. neutral glycans in glycoproteins. Esterification methods have been developed for free sialylated glycans and sialylated Asn-glycans, allowing these acidic groups to ionize with the same efficiency as neutral sugars. METHODS: Here we describe a method which modifies glycopeptides by esterification. This simple procedure is applied to glycopeptides isolated from tryptic digests of monoclonal antibodies (mAbs), some highly sialylated. To better understand the effect of esterification on the peptide backbone, synthetic EEQYNSTYR was esterified and studied by tandem mass spectrometry (MS/MS). Acetamidation of EEQYNSTYR was also studied as some mAb samples had been overalkylated prior to tryptic digestion. RESULTS: As a general trend, ethyl-esterification or lactonization is observed for each sialic acid on glycoforms of EEQYNSTYR (the N-glycosylated tryptic peptide of IgG Fc), depending on the branching position of the sialic acid (α2,3 or α2,6). Esterification also affects the carboxyl groups in the peptide, including the C-terminal COOH. CONCLUSIONS: For antibody analysis, MALDI-MS ion abundances give a better semi-quantitative estimate of sialylation levels for esterified than for unreacted glycopeptides. The method is simple to use and helps to differentiate the branching patterns of sialic acids in antibodies.

Differential acetylcholinesterase activity in rat cerebrum, cerebellum and hypothalamus.

Acetylcholinesterase (AChE) has been purified from three different regions of rat brain using Sephadex G 200 column. SDS PAGE (6%) showed single band for the purified AChE fractions. Purified and lyophilized AChE from different (NH4)2SO4 precipitated fractions of three brain parts were utilized for in vitro enzyme kinetics using Dimethoate (Dmt) as inhibitor. K(m) values for cerebellum and hypothalamus were almost similar whereas cerebrum showed a different K(m) value compared to other two regions. With the drug Rivastigmine it was found that % G1 and G4 forms of AChE in three different parts of brain are different.