Sites Asn Research Articles

Comparison of sialylated and fucosylated N-glycans attached to Asn 6 and Asn 41 with different roles in hyaluronan and proteoglycan link protein 1 (HAPLN1)

Hyaluronan and proteoglycan link protein 1 (HAPLN1) is an extracellular matrix protein stabilizing interactions between hyaluronan and proteoglycan. Although HAPLN1 is being investigated for various biological roles, its N-glycosylation is poorly understood. In this study, the structure of N-glycopeptides of trypsin-treated recombinant human HAPLN1 (rhHAPLN1) expressed from CHO cells were identified by nano-liquid chromatography-tandem mass spectrometry. A total of 66 N-glycopeptides were obtained, including 16 and 12 N-glycans at sites Asn 6 (located in the N-terminal region) and Asn 41 (located in the Ig-like domain, which interacts with proteoglycan), respectively. The quantities (%) of each N-glycan relative to the totals (100 %) at each site were calculated. Tri- and tetra-sialylation (to resist proteolysis and extend half-life) were more abundant at Asn 6, and di- (core- and terminal-) fucosylation (to increase binding affinity and stability) and sialyl-Lewis X/a epitope (a major ligand for E-selectin) were more abundant at Asn 41. These results indicate that N-glycans attached to Asn 6 (protecting HAPLN1) and Asn 41 (supporting molecular interactions) play different roles in HAPLN1. This is the first study of site-specific N-glycosylation in rhHAPLN1, which will be useful for understanding its molecular interactions in the extracellular matrix.

Peptide oxo-esters for ligation of peptide hydrazide at Gln and Asn sites

We describe a practical strategy for ligation of peptide hydrazide at Gln and Asn sites. Peptide hydrazides with Gln, Asn and Asp at the C-terminus would form by-products during TFA-based peptide cleavage due to the intramolecular cyclization of the side chain functional groups with the hydrazide moiety. Therefore, the ligation junctions of Gln-Cys, Asn-Cys and Asp-Cys are rarely chosen for hydrazide-based native chemical ligation. To solve this problem, we propose a strategy for the preparation of peptide hydrazides with C-termnal Gln, Asn and Asp, through the hydrazinolysis of peptide oxo-esters. This strategy avoids the trifluoroacetic acid treatment of peptide hydrazides and can be readily used for the synthesis of peptide hydrazides with C-terminal Gln, Asn and Asp. Through oxidative NaNO2 treatment of peptide hydrazides, we successfully obtain C-terminal thioester peptides of Gln and Asn. The utility of the oxo-ester based strategy has been demonstrated in the chemical synthesis of SHK toxin and is expected to expand the scope of hydrazide-based native chemical ligation in chemical protein synthesis.

The legumain McPAL1 from Momordica cochinchinensis is a highly stable Asx-specific splicing enzyme

Legumains, also known as asparaginyl endopeptidases (AEPs), cleave peptide bonds after Asn/Asp (Asx) residues. In plants, certain legumains also have ligase activity that catalyzes biosynthesis of Asx-containing cyclic peptides. An example is the biosynthesis of MCoTI-I/II, a squash family-derived cyclic trypsin inhibitor, which involves splicing to remove the N-terminal prodomain and then N-to-C-terminal cyclization of the mature domain. To identify plant legumains responsible for the maturation of these cyclic peptides, we have isolated and characterized a legumain involved in splicing, McPAL1, from Momordica cochinchinensis (Cucurbitaceae) seeds. Functional studies show that recombinantly expressed McPAL1 displays a pH-dependent, trimodal enzymatic profile. At pH 4 to 6, McPAL1 selectively catalyzed Asp-ligation and Asn-cleavage, but at pH 6.5 to 8, Asn-ligation predominated. With peptide substrates containing N-terminal Asn and C-terminal Asp, such as is found in precursors of MCoTI-I/II, McPAL1 mediates proteolysis at the Asn site and then ligation at the Asp site at pH 5 to 6. Also, McPAL1 is an unusually stable legumain that is tolerant of heat and high pH. Together, our results support that McPAL1 is a splicing legumain at acidic pH that can mediate biosynthesis of MCoTI-I/II. We purport that the high thermal and pH stability of McPAL1 could have applications for protein engineering.

Haptoglobin as a Biomarker.

—Haptoglobin (Hp) is a glycoprotein that binds free hemoglobin (Hb) in plasma and plays a critical role in tissue protection and prevention of oxidative damage. Besides, it has some regulatory functions. Haptoglobin is an acute-phase protein, its concentration in plasma changes in pathology, and the test for its concentration is part of normal clinical practice. Haptoglobin is a conservative protein synthesized mainly in the liver and lungs and is the subject of research as a potential biomarker of many diseases, including various forms of malignant neoplasms. Haptoglobin has several unique biophysical characteristics. The human Нр gene is polymorphic, has three structural alleles that control the synthesis of three major phenotypes of haptoglobin: homozygous Нр1-1 and Нр2-2, and heterozygous Нр2-1, determined by a combination of allelic variants that are inherited. Numerous studies indicate that the phenotype of haptoglobin can be used to judge the individual predisposition of a person to various diseases. In addition, Hp undergoes various post-translational modifications (PTMs). These are structural transformations (removal of the signal peptide, cutting off the Pre-Hp precursor molecule into two subunits, α and β, limited proteolysis of α-chains, formation of disulfide bonds, multimerization), as well as chemical modifications of α-chains and glycosylation of the β-chain. Glycosylation of the β-chain of haptoglobin at four Asn sites is the most important variable PTM that regulates the structure and function of the glycoprotein. The study of modified oligosaccharides of the β-chain of Hp has become the main direction in the study of pathological processes, including malignant neoplasms. These characteristics indicate the possibility of the existence of Hp in the form of a multitude of proteoforms, probably performing different functions. This review is devoted to the description of the structural and functional diversity and the potential use of Hp as a biomarker of various pathologies.

Haptoglobin as a biomarker

Haptoglobin (Hp) is a blood plasma glycoprotein that binds free hemoglobin (Hb) and plays a critical role in tissue protection and the prevention of oxidative damage. In addition, it has a number of regulatory functions. Haptoglobin is an acute phase protein, its concentration in plasma changes in pathology, and the test for its concentration is part of normal clinical practice. Haptoglobin is a conservative protein synthesized mainly in the liver and lungs and is the subject of research as a potential biomarker of many diseases, including various forms of malignant neoplasms. Haptoglobin has several unique biophysical characteristics. Only in humans, the Hp gene is polymorphic, has three structural alleles that control the synthesis of three major phenotypes of Hp, homozygous Hp1-1 and Hp2-2, and heterozygous Hp2-1, determined by a combination of allelic variants that are inherited. Numerous studies indicate that the phenotype of haptoglobin can be used to judge the individual's predisposition to various diseases. In addition, Hp undergoes various post-translational modifications (PTMs). These are structural transformations (removal of the signal peptide, cutting of the Pre-Hp precursor molecule into two subunits, α and β, limited proteolysis of α-chains, formation of disulfide bonds, multimerization), as well as chemical modifications of α-chains and glycosylation of the β-chain. Glycosylation of the β-chain of haptoglobin at four Asn sites is the most important variable PTM that regulates the structure and function of the glycoprotein. The study of modified oligosaccharides of the Hp β-chain has become the main direction in the study of pathological processes, including malignant neoplasms. Many studies are focused on the identification of PTM and changes in the level of the α2-chain of this protein in pathology. These characteristics of Hp indicate the possibility of the existence of this protein as different proteoforms, probably with different functions. This review is devoted to the description of the structural and functional diversity of Hp and its potential use as a biomarker of various pathologies.

Protein Modifications Critical for Myonectin/Erythroferrone Secretion and Oligomer Assembly.

Myonectin/erythroferrone (also known as CTRP15) is a secreted hormone with metabolic function and a role in stress erythropoiesis. Despite its importance in physiologic processes, biochemical characterization of the protein is lacking. Here, we show that multiple protein modifications are critical for myonectin secretion and multimerization. Abolishing N-linked glycosylation by tunicamycin, glucosamine supplementation, or glutamine substitutions of all four potential Asn glycosylation sites blocked myonectin secretion. Mass spectrometry confirmed that Asn-229 and Asn-281 were glycosylated, and substituting both Asn sites with Gln prevented myonectin secretion. Although Asn-319 is not identified as glycosylated, Gln substitution caused protein misfolding and retention in the endoplasmic reticulum. Of the four conserved cysteines, Cys-273 and Cys-278 were required for proper protein folding; Ala substitution of either site inhibited protein secretion. In contrast, Ala substitutions of Cys-142, Cys-194, or both markedly enhanced protein secretion, suggesting endoplasmic reticulum retention that facilitates myonectin oligomer assembly. Secreted myonectin consists of trimers, hexamers, and high-molecular weight (HMW) oligomers. The formation of higher-order structures via intermolecular disulfide bonds depended on Cys-142 and Cys-194; while the C142A mutant formed almost exclusively trimers, the C194A mutant was impaired in HMW oligomer formation. Most Pro residues within the short collagen domain of myonectin were also hydroxylated, a modification that stabilized the collagen triple helix. Inhibiting Pro hydroxylation or deleting the collagen domain markedly reduced the rate of protein secretion. Together, our results reveal key determinants that are important for myonectin folding, secretion, and multimeric assembly and provide a basis for future structure-function studies.

Engineered ChymotrypsiN for Mass Spectrometry-Based Detection of Protein Glycosylation.

We have engineered the substrate specificity of chymotrypsin to cleave after Asn by high-throughput screening of large libraries created by comprehensive remodeling of the substrate binding pocket. The engineered variant (chymotrypsiN, ChyB-Asn) demonstrated an altered substrate specificity with an expanded preference for Asn-containing substrates. We confirmed that protein engineering did not compromise the stability of the enzyme by biophysical characterization. Comparison of wild-type ChyB and ChyB-Asn in profiling lysates of HEK293 cells demonstrated both qualitative and quantitative differences in the nature of the peptides and proteins identified by liquid chromatography and tandem mass spectrometry. ChyB-Asn enabled the identification of partially glycosylated Asn sites within a model glycoprotein and in the extracellular proteome of Jurkat T cells. ChymotrypsiN is a valuable addition to the toolkit of proteases to aid the mapping of N-linked glycosylation sites within proteins and proteomes.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins.

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Identification of Glycosylation Sites Essential for Surface Expression of the CaVα2δ1 Subunit and Modulation of the Cardiac CaV1.2 Channel Activity

Alteration in the L-type current density is one aspect of the electrical remodeling observed in patients suffering from cardiac arrhythmias. Changes in channel function could result from variations in the protein biogenesis, stability, post-translational modification, and/or trafficking in any of the regulatory subunits forming cardiac L-type Ca(2+) channel complexes. CaVα2δ1 is potentially the most heavily N-glycosylated subunit in the cardiac L-type CaV1.2 channel complex. Here, we show that enzymatic removal of N-glycans produced a 50-kDa shift in the mobility of cardiac and recombinant CaVα2δ1 proteins. This change was also observed upon simultaneous mutation of the 16 Asn sites. Nonetheless, the mutation of only 6/16 sites was sufficient to significantly 1) reduce the steady-state cell surface fluorescence of CaVα2δ1 as characterized by two-color flow cytometry assays and confocal imaging; 2) decrease protein stability estimated from cycloheximide chase assays; and 3) prevent the CaVα2δ1-mediated increase in the peak current density and voltage-dependent gating of CaV1.2. Reversing the N348Q and N812Q mutations in the non-operational sextuplet Asn mutant protein partially restored CaVα2δ1 function. Single mutation N663Q and double mutations N348Q/N468Q, N348Q/N812Q, and N468Q/N812Q decreased protein stability/synthesis and nearly abolished steady-state cell surface density of CaVα2δ1 as well as the CaVα2δ1-induced up-regulation of L-type currents. These results demonstrate that Asn-663 and to a lesser extent Asn-348, Asn-468, and Asn-812 contribute to protein stability/synthesis of CaVα2δ1, and furthermore that N-glycosylation of CaVα2δ1 is essential to produce functional L-type Ca(2+) channels.

Histidine-rich glycoprotein function in hepatocellular carcinoma depends on its N-glycosylation status, and it regulates cell proliferation by inhibiting Erk1/2 phosphorylation.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer mortality. Significantly downregulated histidine-rich glycoprotein (HRG) during the dynamic stages (WB, WB7, and WB11) of neoplastic transformation of WB F344 hepatic oval-like cells was screened out by iTRAQ labeling followed by 2DLC-ESI-MS/MS analysis. HRG expression was significantly lower in HCC tissues. HRG overexpression in Huh7 and MHCC-97H hepatoma cell lines led to decreased cell proliferation, colony-forming ability, and tumor growth, and increased cell apoptosis. HRG could inhibit cell proliferation via the FGF-Erk1/2 signaling pathway by reducing Erk1/2 phosphorylation. On the other hand, the functional expression of HRG was also dependent on the glycosylation status at its N-terminal, especially at the glycosylation site Asn 125. The glycosylation of HRG may play a key competitive role in the interaction between HRG and heparin sulfate for binding bFGF and activating the FGF receptor. These findings provide novel insights into the molecular mechanism of HRG in HCC.

Abstract 5434: A high through-put bioluminescent assay to monitor the deamidation of asparagine and isomerization of aspartate residues in therapeutic proteins and antibodies

Abstract Spontaneous degradation reactions such as oxidation, glycation, deamidation, Isomerization, and racemization are non-enzymatic modifications and produce functionally damaged species reflecting aging effect at the molecular level. L-asparagine (Asn) and L-aspartate (Asp) are among the most unstable residues in proteins- linked to deamidation, isomerization and racemization Rx. Under physiological conditions, succinimide-linked deamidation of Asn occurs with t1/2as short as 6 hrs while that for isoaspartate are generally 10-fold longer. A number of biological peptides and proteins possess labile Asn and Asp residues and the formation of IsoAsp at these sites adversely affect their function. The degradation of Asn and Asp sites via succinimide pathway has significant effect on protein function. Loss of function associated with isoAsp formation are found in many biologically critical proteins and is a major source of antibody instability and micro heterogeneity, Thus it is critical to accurately determine both the levels and the site of Asn deamidation in therapeutic antibodies and proteins. Although mass Spectroscopic platforms have been at the forefront of technologies to quantitate and identify sites of isoAsp formation, it fails in accurately determining Asn level and has problems with Asp isomerization due to the various steps required for sample preparation such as denaturation, reduction, alkylation, and enzyme digestion of the Ab before analysis resulting in problems with Asp isomerization and Asn determination artifacts. Although peptide mapping is the most powerful tool in isoAsp analyses, it is very time consuming and may itself actually cause sample degradation during preparation and analysis and is not always practical when large number of samples are needed for analysis. To improve on and complement existing technologies, we have developed a bioluminescent, homogenous, robust, sensitive and easy to use assay to accurately monitor the deamidation of Asn and isomerization of aspartate. The assay is based on the use of protein isoaspartate methyltransferase to catalyze the methylation of isoaspartate using S-adenosylmethionine (SAM) and resulting in the formation of S-adenosylhomocysteine and methylated isoaspartate. The assay is very sensitive, it can detect as little as 100 fmol of IsoAsp in as little as 5-10 pmol of proteins. Citation Format: Said A. Goueli, Kevin Hsiao, Rushikesh Patel, Vishal Nashine. A high through-put bioluminescent assay to monitor the deamidation of asparagine and isomerization of aspartate residues in therapeutic proteins and antibodies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5434. doi:10.1158/1538-7445.AM2015-5434

Enzymatic Digestion and Mass Spectroscopies of N-Linked Glycans in Lacquer Stellacyanin fromRhus vernicifera

Lacquer stellacyanin was isolated and purified from lacquer acetone powder by continuous Sephadex column chromatographies using Sephadex C-50, DEAE A-50, and C-50 gels. The purified lacquer stellacyanin had a blue color with one major and three minor bands around 26 k Dain SDS PAGE. Trypsin- and chymotrypsin-treated lacquer stellacyanins were examined by LC/MS/MS to determine three N-glycosylation sites (N28, N60, and N102) and were further analyzed by MALDI TOF MS, indicating that the N-linked glycans were attached to the three asparagine (Asn) sites, respectively. In addition, after trypsin digestion and PNGase A and PNGase F treatments to cleave N-linked glycans from the Asn sites, it was found that lacquer stellacyanin had a xylose containing a biantennary N-linked glycan with core fucosylation consisting of 13 sugar residues (a complex type N-linked glycan) by MALDI TOF MS analysis. This is the first report on the structure of an N-linked glycan in lacquer stellacyanin.

Physical Stability Comparisons of IgG1-Fc Variants: Effects of N-Glycosylation Site Occupancy and Asp/Gln Residues at Site Asn 297

The structural integrity and conformational stability of various IgG1-Fc proteins produced from the yeast Pichia pastoris with different glycosylation site occupancy (di-, mono-, and nonglycosylated) were determined. In addition, the physical stability profiles of three different forms of nonglycosylated Fc molecules (varying amino-acid residues at site 297 in the CH 2 domain due to the point mutations and enzymatic digestion of the Fc glycoforms) were also examined. The physical stability of these IgG1-Fc glycoproteins was examined as a function of pH and temperature by high-throughput biophysical analysis using multiple techniques combined with data visualization tools (three index empirical phase diagrams and radar charts). Across the pH range of 4.0-6.0, the di- and monoglycosylated forms of the IgG1-Fc showed the highest and lowest levels of physical stability, respectively, with the nonglycosylated forms showing intermediate stability depending on solution pH. In the aglycosylated Fc proteins, the introduction of Asp (D) residues at site 297 (QQ vs. DN vs. DD forms) resulted in more subtle changes in structural integrity and physical stability depending on solution pH. The utility of evaluating the conformational stability profile differences between the various IgG1-Fc glycoproteins is discussed in the context of analytical comparability studies.

Decellularization followed by PNGase F treatment efficiently removes immunogenic αGal epitopes and other N‐acetylglucosamine structures on the glycocalyx of porcine pulmonary heart valve matrices

BackgroundXenotransplantation leads to hyperacute or acute graft reaction due to interaction of preformed human antibodies, mainly αGal‐antibodies, with carbohydrate structures present on non‐human tissue. By removing these carbohydrate structures from porcine decellularized pulmonary heart valves (PHV), grafts might be generated that allow heart valve replacement therapy as with allogeneic PHV matrices.MethodsThus, after detergents based decellularization (0.5%SDS/0.5%Triton‐X100) cell free PHV matrices were enzymatically treated with α1‐3,6‐galactosidase or PNGase F. The potential impact on the glycocalyx was investigated by histochemical stains utilizing isolectin B4 (IL‐B4), wheat germ agglutinin (WGA), Datura stramonium lectin (DSL), and Ricinus communis agglutinin (RCA I) on decellularized only, and decellularized and enzymatically treated specimens. Native PHV tissue served as controls.ResultsAll used lectins stained native heart valve tissue. Decellularization resulted in reduced IL‐B4 and WGA staining, whereas cell removal had no effect on DSL and RCA I staining. Enzymatic PNGase F treatment resulted in a further decrease of IL‐B4 and WGA staining whereas initially not affected DSL stain was reduced as well. Enzymatic treatment with α1‐3,6‐galactosidase led to a reduction of IL‐B4 stain only.ConclusionDecellularization per se is able to reduce αGal epitopes as demonstrated by IL‐B4 staining. This reduction of αGal epitopes can be enhanced by α1‐3,6‐galactosidase digestion as expected. Enzymatic treatment with PNGase F that recognizes GlcNacβ(1‐N)Asn sites results in removal of carbohydrate structures as αGal and N‐acetyl‐glucosamines to a high degree as demonstrated by IL‐B4, WGA, and DSL stains.In summary, detergent based decellularization followed by PNGase F treatment resembles an efficient way to remove immunogenic epitopes from porcine pulmonary heart valve matrices, thus potentially enabling the generation of xenogeneic PHV matrices for clinical application.

Glycosylation in a Mammalian Expression System Is Critical for the Production of Functionally Active Leukocyte Immunoglobulin-like Receptor A3 Protein

The leukocyte immunoglobulin-like receptor (LILR) A3 is a member of the highly homologous activating and inhibitory receptors expressed on leukocytes. LILRA3 is a soluble receptor of unknown functions but is predicted to act as a broad antagonist to other membrane-bound LILRs. Functions of LILRA3 are unclear primarily because of the lack of high quality functional recombinant protein and insufficient knowledge regarding its ligand(s). Here, we expressed and characterized recombinant LILRA3 (rLILRA3) proteins produced in 293T cells, Escherichia coli, and Pichia pastoris. We found that the purified rLILRA3 produced in the mammalian system was the same size as a 70-kDa native macrophage LILRA3. This is 20 kDa larger than the calculated size, suggesting significant post-translational modifications. In contrast, rLILRA3 produced in E. coli was similar in size to the unprocessed protein, but yeast-produced protein was 2-4 times larger than the unprocessed protein. Treatment with peptide-N-glycosidase F reduced the size of the mammalian cell- and yeast-produced rLILRA3 to 50 kDa, suggesting that most modifications are due to glycosylation. Consistent with this, mass spectrometric analysis of the mammalian rLILRA3 revealed canonical N-glycosylation at the predicted Asn(140), Asn(281), Asn(302), Asn(341), and Asn(431) sites. Functionally, only mammalian cell-expressed rLILRA3 bound onto the surface of monocytes with high affinity, and importantly, only this significantly abrogated LPS-induced TNFα production by monocytes. Binding to monocytes was partially blocked by β-lactose, indicating that optimally glycosylated LILRA3 might be critical for ligand binding and function. Overall, our data demonstrated for the first time that LILRA3 is a potential new anti-inflammatory protein, and optimal glycosylation is required for its functions.

Role of N-glycosylation of human lysosomal phospholipase A2 for the formation of catalytically active enzyme

To understand the role of N-glycosylation of lysosomal phospholipase A2 (LPLA2), four potential N-glycosylation sites in human LPLA2 (hLPLA2) were individually modified replacing asparagine (Asn) with alanine by site-direct mutagenesis. COS-7 cells transiently transfected with wild-type (WT) hLPLA2 gene produced catalytically active LPLA2. A single mutation at 273-, 289-, or 398-Asn partially reduced production of active LPLA2. A single mutation at 99-Asn and quadruple mutations at all four Asn sites resulted in a marked reduction of active LPLA2 and loss of active LPLA2, respectively. Western blot analysis using anti-hLPLA2 antibody showed that the LPLA2 expression level was similar between all transfectants. N-glycosidase F digestion revealed that multiple forms of LPLA2 found in individual transfectants are due to different N-glycans linked to the core protein. The LPLA2 activity in individual transfectants was mostly recovered in the soluble fraction and correlated to the quantity of LPLA2 detected in the soluble fraction. LPLA2 mutated at 99-Asn was mostly retained in the membrane fraction. The WT transfectants treated with tunicamycin markedly lost LPLA2 activity. These data indicate that the 99-Asn is the most critical N-glycosylation site for formation of native hLPLA2 in vivo and that the N-glycosylation of LPLA2 is crucial for biosynthesis of catalytically active hLPLA2.

Accumulation of altered aspartyl residues in erythrocyte membrane proteins from patients with sporadic amyotrophic lateral sclerosis

Spontaneous protein deamidation of labile asparagines (Asn), generating abnormal l-isoaspartyl residues (IsoAsp), is associated with cell aging and enhanced by an oxidative microenvironment. The presence of isopeptide bonds impairs protein structure/function. To minimize the damage, IsoAsp can be “repaired” by the protein l-isoaspartyl/d-aspartyl O-methyltransferase (PIMT) and S-adenosylmethionine (AdoMet) is the methyl donor of this reaction. PIMT is a repair enzyme that initiates the conversion of l-isoAsp (or d-Asp) residues to l-Asp residues. Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease principally affecting motor neurons. The condition of oxidative stress reported in familial and sporadic forms of ALS prompted us to investigate Asn deamidation in ALS tissue. Erythrocytes (RBCs) were selected as a model system since they are unable to replace damaged proteins and protein methylesterification is virtually the only AdoMet-consuming reaction operating in these cells. Our data show that, in vitro assay, abnormal IsoAsp residues were significantly higher in ALS patients erythrocyte membrane proteins with an increased methyl accepting capability relative to controls (p<0.05). Moreover, we observed a reduction in AdoMet levels, while AdoHcy concentration was comparable to that detected in the control, resulting in a lower [AdoMet]/[AdoHcy] ratio. Then, the accumulation of altered aspartyl residues in ALS patients is probably related to a reduced efficiency of the S-adenosylmethionine (AdoMet)-dependent repair system causing increased protein instability at Asn sites. The increase of abnormal residues represents a new protein alteration that may be present not only in red blood cells but also in other cell types of patients suffering from ALS.

Interaction and Inhibition of Dengue Envelope Glycoprotein with Mammalian Receptor DC-Sign, an In-Silico Approach

Membrane fusion is the central molecular event during the entry of enveloped viruses into cells. The critical agents of this process are viral surface proteins, primed to facilitate cell bilayer fusion. The important role of Dendritic-cell-specific ICAM3-grabbing non-integrin (DC-SIGN) in Dengue virus transmission makes it an attractive target to interfere with Dengue virus Propagation. Receptor mediated endocytosis allows the entry of virions due to the presence of endosomal membranes and low pH-induced fusion of the virus. DC-SIGN is the best characterized molecule among the candidate protein receptors and is able to mediate infection with the four serotypes of dengue virus (DENV). Unrestrained pair wise docking was used for the interaction of dengue envelope protein with DC-SIGN and monoclonal antibody 2G12. Pre-processed the PDB coordinates of dengue envelope glycoprotein and other candidate proteins were prepared and energy minimized through AMBER99 force field distributed in MOE software. Protein-protein interaction server, ZDOCK was used to find molecular interaction among the candidate proteins. Based on these interactions it was found that antibody successfully blocks the glycosylation site ASN 67 and other conserved residues present at DC-SIGN-Den-E complex interface. In order to know for certain, the exact location of the antibody in the envelope protein, co-crystallize of the envelope protein with these compounds is needed so that their exact docking locations can be identified with respect to our results.

Modeling and structural analysis of cellulases using Clostridium thermocellum as template.

Cellulase is one of the most widely distributed enzymes with wide application. They are involved in conversion of biomass into simpler sugars. Cellulase of Trichoderma longibrachiatum, a known cellulolytic fungus was compared with Clostridium thermocellum [AAA23226.1] cellulase. Blastp was performed with AAA23226.1 as query sequence to obtain nine similar sequences from NCBI protein data bank. The physicochemical properties of cellulase were analyzed using ExPASy’s ProtParam tool namely ProtParam, SOPMA and GOR IV. Homology modeling was done using SWISS MODEL and checked quality by RMSD values using VMD1.9.1. Active sites of each model were predicted using automated active site prediction server of SCFBio. Study revealed instability of cellulase of two eukaryotic strains namely Trichoderma longibrachiatum [CAA43059.1] and Melanocarpus albomyces [CAD56665.1]. The negative GRAVY score value of cellulases ensured better interaction and activity in aqueous phase. It was found that molecular weight (M. Wt) ranges between 25-127.56 kDa. Iso-electric point (pI) of cellulases was found to be acidic in nature. GOR IV and SOPMA were used to predict secondary structure of cellulase, which showed that random coil, was dominated. Neighbor joining tree with C. thermocellum [AAA23226.1] cellulase as root showed that cellulases of Thermoaerobacter subterraneus [ZP_07835928] and C. thermocellum [CAA4305.1] were more similar to eukaryotic cellulases supported by least boot strap values. Pseudoalteromonas haloplanktis cellulase was found to be the ideal model supported by least RMSD score among the predicted structures. Trichoderma longibrachiatum cellulase was found to be the best compared to other cellulases, which possess high number of active sites with ASN and THR rich active sites. CYS residues were also present ensuring stable interaction and better bonding. Hydrophilic residues were found high in active sites of all analyzed models and template.

Abnormal isoaspartyl residues in erythrocyte membranes from psoriatic patients

Spontaneous protein deamidation of labile asparagines (Asn), generating abnormal isoaspartyl residues (IsoAsp), is associated with cell aging and enhanced by an oxidative microenvironment. The presence of isopeptide bonds impairs protein structure/function and can trigger autoimmune responses. To minimize the damage, IsoAsp can be "repaired" by a specific L-isoaspartate-(D-aspartate)-protein-O-methyltransferase. The condition of chronic oxidative stress reported in psoriatic patients, and the potential etiological role of unknown self-antigens, prompted us to investigate Asn deamidation in psoriatic tissues. Erythrocytes (RBC) were selected as the model system since, lacking protein synthesis apparatus, they are unable to replace damaged proteins. Blood samples were obtained from 36 patients and 34 controls. L-isoAsp content was highly increased in RBC membrane proteins from psoriatic patients. Deamidated species included ankyrin, band 4.1, band 4.2 and the integral membrane protein band 3. A functional analysis demonstrated that this result was unrelated to a reduced efficiency of the S-adenosylmethionine-dependent repair system suggesting an increased protein instability at Asn sites, responsible for IsoAsp accumulation in psoriatic patients.