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BBA - Proteins and Proteomics (v.1774, #3)
Posttranslational modifications in human plasma MBL and human recombinant MBL by Pia Hønnerup Jensen; Inga Laursen; Finn Matthiesen; Peter Højrup / (pp. 335-344).
Mannan-binding lectin (MBL) is a complex serum protein that plays an important role in innate immunity. In addition to assuming several different oligomeric forms, the polypeptide itself is highly heterogeneous. This heterogeneity is due to post-translational modifications, which help to stabilize the intact protein in its active conformation. For the first time, positions and occupation frequency of partial hydroxylations and partial glycosylations are reported in MBL. Hydroxylation and glycosylation patterns of both recombinant and plasma derived MBL were determined, using a combination of mass spectrometry on reduced MBL and on enzyme cleaved MBL. Variations in the degree of hydroxylation and glycosylation seem to be an indigenous characteristic of collectins. In addition to these already known modifications, a new post-translational modification was identified. Cys216 (and occasionally also Cys202) was modified in trace amounts to dehydroalanine, as detected by a 34 Da mass loss. This impairs the formation of a disulphide bond in the carbohydrate recognition domain. The dehydroalanine was identified in similar small amounts in both recombinant and plasma-derived MBL.
Keywords: Abbreviations; MBL; mannan-binding lectin; pMBL; plasma derived human MBL; rhMBL; recombinant human MBL; Hyp; hydroxyproline; DHA; dehydroalanine; CRD; carbohydrate recognition domain; HCCA; α-cyano-4-hydroxycinnamic acid; SA; sinapic acid; DTT; dithiothreitol; TFA; trifluoro acetic acid; MetO; oxidized methionineHydroxylysine; Hydroxyproline; Dehydroalanine from cysteine; Collectin; Mass spectrometry; Plasma protein
Differential susceptibility of alpha A- and alpha B-crystallin to gamma-ray irradiation by Norihiko Fujii; Tooru Nakamura; Yutaka Sadakane; Takeshi Saito; Noriko Fujii (pp. 345-350).
Alpha-crystallin, a major protein of all vertebrate lenses, consists of two different subunits, alpha A and alpha B, which form polymeric aggregates with an average molecular mass of 300-800 kDa. Both the alpha A and alpha B subunit have a molecular mass of about 20 kDa. It is not known why alpha crystallin aggregates comprise two different subunits, given that the physicochemical properties of these proteins are very similar. The present study compares the susceptibility of the alpha A and alpha B subunits to gamma-rays. We prepared a recombinant form of human alpha A- and alpha B-crystallin and then irradiated the proteins with gamma-rays. Based on far-UV CD spectra, alpha A-crystallin retained beta-sheet conformation after gamma irradiation up to 3.0 kGy, whereas alpha B-crystallin lost beta-sheet conformation upon exposure to gamma irradiation at >1.0 kGy. Size exclusion chromatography showed that the aggregation and polydispersity of recombinant alpha A-crystallin increased slightly after >1.0 kGy irradiation. In contrast, irradiation of alpha B-crystallin at 1.0 kGy resulted in the formation of huge aggregates and a marked increase in heterogeneity. We have also compared the chaperone activities of gamma-irradiated alpha A- and alpha B-crystallin aggregates. The activity of irradiated alpha A-crystallin was retained while that of the irradiated alpha B-crystallin was became inactive after irradiation of >0.5 kGy. Our results indicate that alpha A-crystallin is more stable to gamma irradiation than alpha B-crystallin.
Keywords: Alpha A-crystallin; Alpha B-crystallin; Gamma-ray; Lens; Oxidation
Secondary structure analysis of HIV-1-gp41 in solution and adsorbed to aluminum hydroxide by Fourier transform infrared spectroscopy by Audrey Agopian; Frédéric Ronzon; Elisabeth Sauzéat; Régis Sodoyer; Raphaelle El Habib; René Buchet; Michel Chevalier (pp. 351-358).
The formulation of human vaccines often includes adjuvants such as aluminum hydroxide that are added to enhance the immune responses to vaccine antigens. However, these adjuvants may also affect the conformation of antigenic proteins. Such structural modifications could lead to changes in antigenicity such that suboptimal protective immune responses could be generated relative to those induced by the vaccine antigens alone. Here, we used attenuated total reflectance infrared spectroscopy (ATR-FTIR) to compare the secondary structures of recombinant HIV-1-gp41 (gp41) in solution or adsorbed to aluminum hydroxide. The gp41 secondary structure content was 72% α-helices and 28% β-sheets in 5 mM formate buffer p2H 2.5, while it was 66% β-sheets and 34% random coil in acetonitril/2H2O (95/5:v/v). A fully reversible conformational change of gp41 in acetonitril/2H2O (95/5:v/v) was observed upon addition of either 35 mM formate p2H 2.5 or 0.1% (w/v) detergent (Tween 20, Hecameg, Brij 35 or β-d-octyl-glucopyranoside). When gp41 was adsorbed to aluminum hydroxide in the presence of 0.1% (w/v) detergent, in either formate or in acetonitril/2H2O (95/5:v/v) its secondary structure remained stable and was identical to that of gp41 in 5 mM formate buffer p2H 2.5. The method described here could be applied for the characterization of gp41 conformers for use in immunological screening of antigens, and more generally to all antigenic proteins adsorbed to aluminum hydroxide.
Keywords: Infrared spectroscopy; Alum; HIV1-gp41
Mechanism of flavin reduction in the alkanesulfonate monooxygenase system by Benlian Gao; Holly R. Ellis (pp. 359-367).
The alkanesulfonate monooxygenase system from Escherichia coli is involved in scavenging sulfur from alkanesulfonates under sulfur starvation. An FMN reductase (SsuE) catalyzes the reduction of FMN by NADPH, and the reduced flavin is transferred to the monooxygenase (SsuD). Rapid reaction kinetic analyses were performed to define the microscopic steps involved in SsuE catalyzed flavin reduction. Results from single-wavelength analyses at 450 and 550 nm showed that reduction of FMN occurs in three distinct phases. Following a possible rapid equilibrium binding of FMN and NADPH to SsuE (MC-1) that occurs before the first detectable step, an initial fast phase (241 s−1) corresponds to the interaction of NADPH with FMN (CT-1). The second phase is a slow conversion (11 s−1) to form a charge–transfer complex of reduced FMNH2 with NADP+ (CT-2), and represents electron transfer from the pyridine nucleotide to the flavin. The third step (19 s−1) is the decay of the charge–transfer complex to SsuE with bound products (MC-2) or product release from the CT-2 complex. Results from isotope studies with [(4 R)-2H]NADPH demonstrates a rate-limiting step in electron transfer from NADPH to FMN, and may imply a partial rate-limiting step from CT-2 to MC-2 or the direct release of products from CT-2. While the utilization of flavin as a substrate by the alkanesulfonate monooxygenase system is novel, the mechanism for flavin reduction follows an analogous reaction path as standard flavoproteins.
Keywords: Abbreviations; SsuE; alkanesulfonate FMN reductase; SsuD; alkanesulfonate monooxygenase; Fre; NAD(P)H:flavin reductase; CT; charge–transfer complex; MC; Michaelis complex; FNR; ferredoxin-NADP; +; reductaseAlkanesulfonate monooxygenase; FMN reductase; SsuE; SsuD; Charge–transfer complex; Rapid reaction kinetics
The human urine mannose 6-phosphate glycoproteome by David E. Sleat; Haiyan Zheng; Peter Lobel (pp. 368-372).
Glycoproteins containing the mannose 6-phosphate (Man-6-P) modification represent a class of proteins of considerable biomedical importance. They include over sixty different soluble lysosomal hydrolases and accessory proteins, deficiencies of which result in over forty different known human genetic diseases. In addition, there are patients with lysosomal storage diseases of unknown etiology and lysosomal proteins have been implicated in pathophysiological processes associated with Alzheimer disease, arthritis, and cancer. The aim of this study was to explore urine as a source for the proteomic investigation of lysosomal storage disorders as well as for biomarker studies on the role of Man-6-P containing proteins in other human diseases. To this end, urinary proteins were affinity purified on immobilized Man-6-P receptors, digested with trypsin, and analyzed using nanospray LC/MS/MS. This resulted in identification of 67 proteins, including 48 known lysosomal proteins and 9 proteins that may be lysosomal. The identification of a large proportion of the known set of soluble lysosomal proteins with relatively few contaminants suggests that urine represents a promising substrate for the development of comparative proteomic methods for the investigation of lysosomal disorders and other diseases involving Man-6-P glycoproteins.
Keywords: Human urine; Mannose-6 phosphate; Lysosome; Lysosomal enzyme; Proteomic; Mass spectrometry
Insight into the virulence of Rickettsia prowazekii by proteomic analysis and comparison with an avirulent strain by Chien-Chung Chao; Dirk Chelius; Terry Zhang; Elissa Mutumanje; Wei-Mei Ching (pp. 373-381).
Rickettsia prowazekii, an obligate intracellular Gram-negative bacterium, is the etiologic agent of epidemic typhus. We analyzed the proteome of the virulent Breinl strain of R. prowazekii purified from infected egg yolk sacs. Total proteins from purified R. prowazekii Breinl strain were reduced by dithiothreitol, alkylated by iodoacetic acid and digested with trypsin followed by analysis with an integrated two-dimensional liquid chromatography and mass spectrometry system (2D-LC/MS/MS). A comparison was made using previously analyzed proteome of the Madrid E strain and current analysis of the Breinl strain. For Breinl 251 proteins were identified, representing 30% of the total protein-encoding genes, using a shotgun 2D-LC/MS/MS proteomic approach. This result is identical to that of Madrid E strain. Among the identified proteins, 33 from Breinl and 37 from Madrid E have an unknown function. A methyltransferase, RP028/RP027, whose gene is mutated in the avirulent Madrid E strain but not in the virulent Breinl strain, was only detectable in the Breinl strain, consistent with the genetic mutation in Madrid E. This result suggests the possible relationship between this gene product and the virulence of the strains.
Keywords: Rickettsia prowazekii; Proteomics; LC-MS-MS; Different abundance; Virulent
Conformational study of palindromic tripeptides (GPG, IPI and KPK) in HIV-1 protease—A density functional theory study by A. Abiram; P. Kolandaivel (pp. 382-391).
A comparative study has been carried out on three palindromic tripeptides Gly–Pro–Gly, Ile–Pro–Ile and Lys–Pro–Lys which were present in HIV protein along with their analogues applying density functional computation at B3LYP/6-31G* level of theory. Discrepancy from the structural analysis has been noted for all the systems and it was found to be more for amide capped structure at the C terminal of proline. The puckering amplitude A and Phase angle P of the pyrrolidine ring of proline in the chosen palindromic tripeptides and their analogues were calculated from the endocyclic torsion angles. The minimum energy conformers lying well within the prescribed region of proline were obtained for the derived compounds from potential energy surface scan mentioning that no role has been played by its terminal residues. This is further supported by the simulated amide bands identifying the helical structure for all three palindromic tripeptides signifying the importance of proline. The molecular properties such as stabilization energy, chemical hardness along with dipole moment were calculated and interpreted. The values of Cα-Hs and the peptide backbone N–Cα–CO for all the selected conformers specify the three palindromic tripeptides to have a symmetrical achiral structure.
Keywords: Abbreviations; GPG; Glycine–Proline–Glycine; IPI; Isoleucine–Proline– Isoleucine; KPK; Lysine–Proline–Lysine; HIV; Human Immuno Virus; H; s; The side chain hydrogen atom in proline; PES; Potential Energy Surface; A; Amplitude of pucker; P; Phase angle of the pyrrolidine ring of proline; Boc; Tetra butyl oxy carbonyl; χ; i; 0; , χ; i; 1; , χ; i; 2; , χ; i; 3; and χ; i; 4; Endocyclic torsion angles of the five membered ring in prolineDensity functional theory; Proline; Amide capping; Pseudorotation; PES
Purification and physico-kinetic characterization of 3β-hydroxy specific sterol glucosyltransferase from Withania somnifera (L) and its stress response by Bhaskara Reddy Madina; Lokendra Kumar Sharma; Pankaj Chaturvedi; Rajender Singh Sangwan; Rakesh Tuli (pp. 392-402).
Sterol glycosyltransferases catalyze the synthesis of diverse glycosteroids in plants, leading to a change in their participation in cellular metabolism. Withania somnifera is a medically important plant, known for a variety of pharmacologically important withanolides and their glycosides. In this study, a cytosolic sterol glucosyltransferase was purified 3406 fold to near homogeneity from W. somnifera leaves and studied for its biochemical and kinetic properties. The purified enzyme was active with UDP-glucose but not with UDP-galactose as sugar donor. It exhibited broad sterol specificity by glucosylating a variety of sterols and phytosterols with 3β-OH group. It showed a low level of activity with flavonoids and isoflavonoids. The enzyme gave maximum Kcat/ Km value (0.957) for 24-methylenecholesterol that resembles aglycone structure of pharmacologically important sitoindosides VII and VIII from W. somnifera. The enzyme follows ordered sequential bisubstrate mechanism of reaction, in which UDP-glucose and sterol are the first and second binding substrates. This is the first detailed kinetic study on purified plant cytosolic sterol glucosyltransferases. Results on peptide mass fingerprinting and substrate specificity suggested that the enzyme belongs to the family of secondary metabolite glucosylating glucosyltransferases. The enzyme activity exhibited a rapid in vivo response to high temperature and salicylic acid treatment of plants, suggesting its physiological role in abiotic and biotic stress.
Keywords: Heat stress; Salicylic acid signal; Sterol glucoside; Sterol glucosyltransferase; Stress response; Substrate specificity; Withanosides
ANS fluorescence: Potential to augment the identification of the external binding sites of proteins by Oktay K. Gasymov; Ben J. Glasgow (pp. 403-411).
8-anilino-1-naphthalenesulfonic acid (ANS) is believed to strongly bind cationic groups of proteins and polyamino acids through ion pair formation. A paucity of data exists on the fluorescent properties of ANS in these interactions. ANS binding to arginine and lysine derivatives was studied by fluorescence and circular dichroism spectroscopies to augment published information attained by isothermal titration calorimetry (ITC). Fluorescence enhancement with a hypsochromic shift results from the interaction of the charged group of lysine and arginine with the sulfonate group of ANS. Ion pairing between Arg (or Lys) and the sulfonate group of ANS reduce the intermolecular charge transfer (CT) rate constant that leads to enhancement of fluorescence. A positive charge near the –NH group of ANS changes the intramolecular CT process producing a blue shift of fluorescence. The Arg side chain compared to that of Lys more effectively interacts with both the –NH and sulfonate groups of ANS. ANS binding also induces a random coil-alpha helix transition in poly-Arg. Our data, in contrast to ITC results, indicate that electrostatic interactions between ANS derivatives and positively charged side chains do not account for binding affinity in the micromolar range. In addition to ion pairing complementary interactions, such as van der Waals, should be considered for high affinity ( Kd<1 mM) external binding sites of proteins.
Keywords: ANS fluorescence; External binding site; Fluorescence lifetime; Circular dichroism; Polyamino acid; Competitive binding assay; Hydrophobic probe; Random coil-alpha helix transition; Ion pairing mechanism; Intramolecular charge transfer