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PubMed2002

Art v 1, the major allergen of mugwort pollen, is a modular glycoprotein with a defensin-like and a hydroxyproline-rich domain.

In late summer, pollen grains originating from Compositae weeds (e.g., mugwort, ragweed) are a major source of allergens worldwide. Here, we report the isolation of a cDNA clone coding for Art v 1, the major allergen of mugwort pollen. Sequence analysis showed that Art v 1 is a secreted allergen with an N-terminal cysteine-rich domain homologous to plant defensins and a C-terminal proline-rich region containing several (Ser/Ala)(Pro)2-4 repeats. Structural analysis showed that some of the proline residues in the C-terminal domain of Art v 1 are posttranslationally modified by hydroxylation and O-glycosylation. The O-glycans are composed of 3 galactoses and 9-16 arabinoses linked to a hydroxyproline and represent a new type of plant O-glycan. A 3-D structural model of Art v 1 was generated showing a characteristic "head and tail" structure. Evaluation of the antibody binding properties of natural and recombinant Art v 1 produced in Escherichia coli revealed the involvement of the defensin fold and posttranslational modifications in the formation of epitopes recognized by IgE antibodies from allergic patients. However, posttranslational modifications did not influence T-cell recognition. Thus, recombinant nonglycosylated Art v 1 is a good starting template for engineering hypoallergenic vaccines for weed-pollen therapy.

Allergens↗
PubMed1984

Posttranslational protein modification by amino acid addition in intact and regenerating axons of the rat sciatic nerve.

Experiments were performed to determine whether posttranslational addition of amino acids to axonal proteins occurs in axons of the rat sciatic nerve. Two ligatures were placed 1 cm apart on sciatic nerves. Six days later, segments proximal to each ligature were removed, homogenized, centrifuged at 150,000 X g, and analyzed for the ability to incorporate 3H-amino acids into proteins. No incorporation of amino acids into proteins was found in the high-speed supernatant, but when the supernatant was passed through a Sephacryl S-200 chromatography column (removing molecules less than 20 kD), [3H]arginine, lysine, leucine and aspartic acid were incorporated into proteins in both proximal and distal nerve segments. Small but consistently greater amounts of radioactivity were incorporated into proteins in proximal segments compared with distal segments, indicating that the components necessary for the reaction are transported axonally. This reaction represents the posttranslational incorporation of a variety of amino acids into proteins of rat sciatic nerve axons. Other experiments showed that the incorporation of amino acids into proteins is by covalent bonding, that the amino acid donor is likely to be tRNA, and that the reaction is inhibited in vivo by a substance whose molecular mass is less than 20 kD. This inhibition is not affected by incubation with physiological concentrations of unlabeled amino acids, by boiling, or by treatment with Proteinase K. When the axonally transported component of the reaction was determined in regenerating nerves, the amount of incorporation of amino acids into protein was 15-150 times that in intact nerves.(ABSTRACT TRUNCATED AT 250 WORDS)

Acylation↗
PubMed1995

Expression of the Neural Cell Adhesion Molecule NCAM by Peptide- and Steroid-Producing Endocrine Cells and Tumors: Alternatively Spliced Forms and Polysialylation.

The adhesive properties of neural cell adhesion molecules (NCAMs) can be modified by alternative splicing of the primary transcript or by posttranslational modifications, such as sialylation. In this article, we describe distinct forms of alternative splicing and posttranslational modification of the extracellular domain of NCAM of various endocrine tissues and derived tumor cells of the rat and of steroid- and peptide-hormone producing endocrine cells in humans. NCAM-140 is the major isoform expressed in the rat adrenal gland, adenohypophysis, and in granulosa and granulosa-lutein cells. NCAM-180 is predominant in the neurohypophysis. Polysialylated NCAM is expressed in different endocrine tissues and tumor cells of the rat. Different amounts of NCAM mRNA containing the "extra-exon" VASE at the exon 7/8 splice boundary were detected in endocrine cells of rats. Human granulosa cells in culture undergo luteinization. During this process, the VASE-containing NCAM isoform is supplemented by an alternatively spliced isoform without this insert. Thus, modifications of NCAM may be important for adhesive interactions in normal and neoplastic endocrine cells. In addition, the differential expression and the alternative splicing of NCAM during luteinization of granulosa cells raise the possibility that NCAM could be involved in folliculogenesis and the formation of the corpus luteum in humans.

Journal Article↗
PubMed1997

Nonlipopolysaccharide surface antigens of Campylobacter species.

Among the protein antigens of Campylobacter species, flagellin, the subunit of the flagellar filament, is the best characterized. The motility imparted by this locomotory organelle is absolutely essential for Campylobacter organisms to colonize the gastrointestinal tract and to cause diarrheal disease. Flagellin is the immunodominant protein recognized during infection and has been suggested to be involved in the protective immune response. Campylobacter flagellins are glycosylated, which is an unusual posttranslational modification for prokaryotic proteins. Although the chemical structure of the glycosylated moiety is undetermined, the posttranslational modification includes sialic acid. The association of glycosylated flagellin with development of Guillain-Barré syndrome remains speculative, but the possibility of molecular mimicry between glycosylated flagellin and eukaryotic glycoproteins exists.

Antigens, Bacterial↗
PubMed2000

An Assessment of alphaalpha-Tubulin Isotype Modification in Developing Cotton Fiber.

Multiple isoforms of alpha- and beta-tubulin accumulate in higher plant cells, including cotton (Gossypium hirsutum L.) fiber. Isotypes may originate either by transcription of distinct genes or by posttranslational modification of gene products. The existence of two types of posttranslational modification in cotton fiber alpha-tubulin has been examined by immunoblotting cotton fiber proteins from two developmental stages and by probing with specific monoclonal antibodies to acetylated (6-11B-1) or tyrosinated (YL 1/2 ) alpha-tubulin. Control experiments were conducted with an antibody (YOL 1/34) that recognizes a conserved region in plant and animal alpha-tubulins. No acetylated forms of alpha-tubulin were found in either of two varieties of cotton fiber at 10 or 20 d postanthesis. One isotype of alpha-tubulin, isotype 6, failed to cross-react with YL 1/2 at 10 d postanthesis, which indicated that the protein was detyrosinated, and isotype 8 appeared to be detyrosinated at 20 d postanthesis. Since the carboxyl terminus of higher plant alpha-tubulin is exposed on the surface of microtubules, removal of the carboxyl-terminal amino acid may lead to some of the changes in the structure and organization of microtubules that are associated with fiber development.

Journal Article↗
PubMed2005

Biological functions of biotinylated histones.

Histones H1, H2A, H2B, H3 and H4 are DNA-binding proteins that mediate the folding of DNA into chromatin. Various posttranslational modifications of histones regulate processes such as transcription, replication and repair of DNA. Recently, a novel posttranslational modification has been identified: covalent binding of the vitamin biotin to lysine residues in histones, mediated by biotinidase and holocarboxylase synthetase. Here we describe a novel peptide-based technique, which was used to identify eight distinct biotinylation sites in histones H2A, H3 and H4. Biotinylation site-specific antibodies were generated to investigate biological functions of histone biotinylation. Evidence was provided that biotinylation of histones plays a role in cell proliferation, gene silencing and cellular response to DNA damage.

Animals↗
PubMed2001

Broadband detection electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry to reveal enzymatically and chemically induced deamidation reactions within peptides.

Among the numerous forms of chemical degradation of peptides or proteins, deamidation is one of the alterations observed most frequently. In this irreversible reaction, a glutamine or an asparagine side chain is hydrolyzed to glutamic acid or aspartic acid, respectively (conversion of NH2 to OH). Besides its influence in the deterioration of biotechnological and food products, deamidation represents a defined posttranslational modification reaction with respect to proteomics. Here mass spectrometric techniques play a leading role in determining posttranslational modifications. However, not all mass spectrometers are able to resolve signal differences of 0.0193 Da (mass difference of 12CO vs 13CNH) for singly charged molecules, the mass difference between the first isotopic signal of an asparagine/glutamine-containing peptide and the monoisotopic signal of the corresponding partially deamidated aspartate/glutamate derivative. To detect partial deamidation within peptides, advantage has been taken of the ability of Fourier transform ion cyclotron resonance mass spectrometry to perform very high mass resolution. In this work, we investigated up to triply charged ions produced by electrospray ionization using direct infusion. Although the special heterodyne detection mode enables higher mass resolution than the routinely used broadband detection, often only a small mass window can be investigated. Using broadband detection, we were able to resolve ions with a difference of m/z 0.0064 to detect partially deamidated peptides formed either enzymatically or under acidic and basic conditions.

Amides↗
PubMed2005

InsPecT: identification of posttranslationally modified peptides from tandem mass spectra.

Reliable identification of posttranslational modifications is key to understanding various cellular regulatory processes. We describe a tool, InsPecT, to identify posttranslational modifications using tandem mass spectrometry data. InsPecT constructs database filters that proved to be very successful in genomics searches. Given an MS/MS spectrum S and a database D, a database filter selects a small fraction of database D that is guaranteed (with high probability) to contain a peptide that produced S. InsPecT uses peptide sequence tags as efficient filters that reduce the size of the database by a few orders of magnitude while retaining the correct peptide with very high probability. In addition to filtering, InsPecT also uses novel algorithms for scoring and validating in the presence of modifications, without explicit enumeration of all variants. InsPecT identifies modified peptides with better or equivalent accuracy than other database search tools while being 2 orders of magnitude faster than SEQUEST, and substantially faster than X!TANDEM on complex mixtures. The tool was used to identify a number of novel modifications in different data sets, including many phosphopeptides in data provided by Alliance for Cellular Signaling that were missed by other tools.

Databases, Factual↗
PubMed1997

Structure of the carboxy-terminal fragment of the apo-biotin carboxyl carrier subunit of Escherichia coli acetyl-CoA carboxylase.

The biotin carboxyl carrier protein (BCCP) is a subunit of acetyl-CoA carboxylase, a biotin-dependent enzyme that catalyzes the first committed step of fatty acid biosynthesis. In its functional cycle the biotin carboxyl carrier protein engages in heterologous protein-protein interactions with three distinct partners, depending on its state of posttranslational modification. Apo-BCCP interacts specifically with the biotin holoenzyme synthetase, BirA, which results in the posttranslational attachment of biotin to an essential lysine residue on BCCP. Holo-BCCP then interacts with the biotin carboxylase subunit, which leads to the addition of the carboxylate group of bicarbonate to biotin. Finally, the carboxybiotinylated form of BCCP interacts with transcarboxylase in the conversion of acetyl-CoA to malonyl-CoA. The determinants of protein-protein interaction specificity in this system are unknown. One hypothesis is that posttranslational modification of BCCP may result in conformational changes that regulate specific protein-protein interactions. To test this hypothesis, we have determined the NMR solution structure of the unbiotinylated form of an 87 residue C-terminal domain fragment of BCCP (apoBCCP87) from Escherichia coli acetyl-CoA carboxylase and compared this structure with the high-resolution structure of the biotinylated form that was recently solved by X-ray crystallographic techniques. Although the overall folding of the two proteins is highly similar, small structural differences are apparent for residues of the biotin-binding loop that may be important for mediating specific protein-protein interactions.

Acetyl-CoA Carboxylase↗
PubMed1997

The conversion of fibrinogen to fibrin: recombinant fibrinogen typifies plasma fibrinogen.

Plasma fibrinogen is a mixture of multiple molecular forms arising mainly through alternative mRNA processing and subsequent posttranslational modification. Recombinant fibrinogen is synthesized without alternative mRNA processing in a cultured cell system that may generate novel posttranslational modifications. Thus, to show that recombinant fibrinogen can serve as a functional model for plasma fibrinogen, we have examined the conversion of fibrinogen to fibrin, comparing the recombinant with the plasma protein. We examined the kinetics of (1) thrombin-catalyzed fibrinopeptide release, (2) thrombin-catalyzed polymerization of fibrinogen, (3) the polymerization of fibrin monomers, and (4) FXIIIa-catalyzed cross-link formation. We saw small differences in polymerization, suggesting that the ordered assembly of protofibrils and fibers was not identical. In all other analyses, we found that plasma fibrinogen and recombinant fibrinogen were remarkably similar. Using electron microscopy, we examined the structures of individual fibrinogen molecules and fibrin clots. Individual fibrinogen molecules were predominantly three nodule structures for both recombinant and plasma proteins. Both samples also displayed four nodule structures, but fewer four nodule structures were found with recombinant fibrinogen. Fibrin clot structures were essentially indistinguishable. We concluded that recombinant fibrinogen can serve as a accurate model for plasma fibrinogen.

Animals↗
PubMed1996

Effect of the UV modification of alpha-crystallin on its ability to suppress nonspecific aggregation.

Recent studies have shown that structural modifications of alpha-crystallin during lens aging decrease it's effectiveness as a molecular chaperone. Some of these posttranslational modifications have been linked to UV radiation, and this study was undertaken to investigate the effect of UV irradiation on the ability of alpha-crystallin to suppress nonspecific aggregation. The effect of 3-hydroxykynurenine (3-HK) was also investigated as a model for its glucoside (3-HKG), a main lens chromophore that has been linked to photochemical changes in the human lens. Alpha- and gamma-crystallin solutions (1 mg/mL, 1:0.125 wt/wt) were photolyzed (transmission above 295 nm) for various time intervals. Thermal denaturation of gamma-crystallin with or without alpha-crystallin was carried out at 70 degrees C and increases in light scattering were measured at 360b nm. We found that (1) irradiation of gamma-crystallin increased its susceptibility to heat-induced scattering. The addition of alpha-crystallin protects it against thermal denaturation, although its ability to do so decreases the longer gamma-crystallin is irradiated and (2) irradiation of alpha-crystallin decreases its ability to suppress nonspecific aggregating and the presence of of 3-HK during irradiation decreases it further. Our results indicate that posttranslational modifications of alpha-crystallin due to UV irradiation affect the sites and mechanisms by which it interacts with gamma-crystallin. The kinetics of gamma-crystallin unfolding during thermal denaturation were also analyzed. We found that a simple two state model applies for nonirradiated gamma-crystallin. This model does not hold when gamma-crystallin is irradiated in the presence or absence of alpha-crystallin. In these cases, two step or multistep mechanisms are more likely.

Animals↗
PubMed2000

Histone deacetylases, transcriptional control, and cancer.

A key event in the regulation of eukaryotic gene expression is the posttranslational modification of nucleosomal histones, which converts regions of chromosomes into transcriptionally active or inactive chromatin. The most well studied posttranslational modification of histones is the acetylation of epsilon-amino groups on conserved lysine residues in the histones' amino-terminal tail domains. Significant advances have been made in the past few years toward the identification of histone acetyltransferases and histone deacetylases. Currently, there are over a dozen cloned histone acetyltransferases and at least eight cloned human histone deacetylases. Interestingly, many histone deacetylases can function as transcriptional corepressors and, often, they are present in multi-subunit complexes. More intriguing, at least some histone deacetylases are associated with chromatin-remodeling machines. In addition, several studies have pointed to the possible involvement of histone deacetylases in human cancer. The availability of the cloned histone deacetylase genes has provided swift progress in the understanding of the mechanisms of deacetylases, their role in transcription, and their possible role in health and disease.

Animals↗
PubMed1983

Chlamydomonas alpha-tubulin is posttranslationally modified in the flagella during flagellar assembly.

The principal alpha-tubulin within Chlamydomonas reinhardtii flagellar axonemes differs from the major alpha-tubulin in the cell body. We show that these two isoelectric variants of alpha-tubulin are related to one another since posttranslational modification of the cell body precursor form converts it to the axonemal form. During flagellar assembly, precursor alpha-tubulin enters the flagella and is posttranslationally modified within the flagellar matrix fraction prior to or at the time of its addition to the growing axonemal microtubules. Experiments designed to identify the nature of this posttranslational modification have also been conducted. When flagella are induced to assemble in the absence of de novo protein synthesis, tritiated acetate can be used to posttranslationally label alpha-tubulin in vivo and, under these conditions, no other flagellar polypeptides exhibit detectable labeling.

Chlamydomonas↗
PubMed2000

An early step of glycosylphosphatidyl-inositol anchor biosynthesis is abolished in lepidopteran insect cells following baculovirus infection.

The expression of recombinant proteins in their native state has become a prerequisite for a variety of functional and structural studies, as well as vaccine development. Many biochemical properties and functions of proteins are dependent on or reside in posttranslational modifications, such as glycosylation. The baculovirus system has increasingly become the system of choice due to it capabilities of performing posttranslational modifications and usually high yields of recombinant proteins. The Toxoplasma gondii surface antigen SAG1 was used as a model for a glycosylphosphatidyl-inositol (GPI)-anchored protein and expressed in insect cells using the baculovirus system. We show that the T. gondii SAG1 surface antigen expressed in this system was not modified by a GPI-anchor. In vitro and in vivo studies demonstrate that uninfected insect cells are able to produce GPI-precursors and to transfer a mature GPI-anchor to nascent proteins. These cells however are not capable to produce GPI-precursors following infection. We also show that the biosynthesis of the early GPI intermediate GlcNH(2)-PI is blocked in baculovirus-infected H5 cells, thus preventing the subsequent mannosylation steps for the synthesis of the conserved GPI-core-glycan. We therefore conclude that the baculovirus system is not appropriate for the expression of GPI-anchored proteins.

Animals↗
PubMed1996

Spiralin polymorphism in strains of Spiroplasma citri is not due to differences in posttranslational palmitoylation.

Spiralin is defined as the major membrane protein of the helical mollicute Spiroplasma citri. According to the S. citri strain used, spiralin shows polymorphism in its electrophoretic mobility. The spiralin gene sequences of eight S. citri strains were determined by direct sequencing of the PCR-amplified genes. All spiralins were found to be 241 amino acids long, except for the spiralin of strain Palmyre, which is 242 amino acids long. The molecular masses calculated from these sequences did not explain the differences observed in the electrophoretic mobilities. In all of the spiralins examined, the first 24 N-terminal amino acids were conserved, including a cysteine at position 24, and had the features of typical signal peptides of procaryotic lipoproteins. When S. citri strains were grown in the presence of [3H]palmitic acid, at least 10 proteins, including spiralin, became labeled. In the presence of globomycin, a lipoprotein signal peptidase inhibitor in eubacteria, apparently unprocessed spiralin could be detected. Formic acid hydrolysis of the [3H]palmitic acid-labeled spiralins of four representative S. citri strains yielded two peptide fragments for each spiralin, as expected from the gene sequence. On fragment was [3H]palmitic acid labeled, and it had almost the same electrophoretic mobility irrespective of the spiralins used. Samples of the unlabeled peptide fragments from the four representative strains had slightly different electrophoretic mobilities (delta Da approximately equal to 800 Da); however, these were much smaller than those of the whole spiralins before formic acid hydrolysis (delta Da approximately equal to 8,000 Da). These results suggest that spiralin polymorphism in S. citri is not due to differences in posttranslational modification by palmitic acid and is certainly a structural property of the whole protein or could result from an unidentified posttranslational modification of spiralin.

Amino Acid Sequence↗
PubMed2006

The Heterochromatin Protein 1 family.

Heterochromatin Protein 1 (HP1) was first discovered in Drosophila as a dominant suppressor of position-effect variegation and a major component of heterochromatin. The HP1 family is evolutionarily conserved, with members in fungi, plants and animals but not prokaryotes, and there are multiple members within the same species. The amino-terminal chromodomain binds methylated lysine 9 of histone H3, causing transcriptional repression. The highly conserved carboxy-terminal chromoshadow domain enables dimerization and also serves as a docking site for proteins involved in a wide variety of nuclear functions, from transcription to nuclear architecture. In addition to heterochromatin packaging, it is becoming increasingly clear that HP1 proteins have diverse roles in the nucleus, including the regulation of euchromatic genes. HP1 proteins are amenable to posttranslational modifications that probably regulate these distinct functions, thereby creating a subcode within the context of the 'histone code' of histone posttranslational modifications.

Animals↗
PubMed1998

A role for amyloid precursor-like protein 2 in corneal epithelial wound healing.

PURPOSE: The authors previously showed that the mRNA encoding the amyloid precursor-like protein 2 (APLP2) was one of several genes with upregulated expression in healing corneal epithelium. To elucidate the physiological role of APLP2 in corneal epithelial cells, the expression, distribution, and posttranslational modification of APLP2 were investigated. METHODS: Alternative splicing was assessed by reverse transcription-polymerase chain reaction; translational expression and posttranslational modification were assessed by chondroitinase digestion and Western blotting. The differential distribution of APLP2 in corneal epithelium was examined by in situ hybridization and immunohistochemical analyses. RESULTS: In rat corneal epithelium, the majority of APLP2 mRNA generated from two alternative splicing sites was found to contain the exon encoding a Kunitz protease inhibitor domain in the first site but to lack the exon encoding a 12-amino acid insert in the second site. The absence of the 12-amino acid insert indicated that APLP2 could be modified by the addition of a chondroitin sulfate (CS) glycosaminoglycan chain. The CS proteoglycan nature of APLP2 was verified by chondroitinase digestion. After wounding, APLP2 mRNA and polypeptides were increased markedly in the basal epithelial cells that were actively migrating. Furthermore, APLP2 was observed in the denuded wound bed immediately adjacent to the leading edge of migratory cells and under the epithelial sheet after wound closure. CONCLUSIONS: The wound-induced, basal-cell-specific APLP2 expression correlates with epithelial cell migration. The spatial and temporal expression of Kunitz protease inhibitor-containing, CS-modified APLP2 in healing corneal epithelium is consistent with its hypothesized role(s) in mediating reorganization of the extracellular matrix and dynamic cell-matrix adhesion during reepithelialization.

Alternative Splicing↗
PubMed2005

Absolute quantitation of cancer-related proteins using an MS-based peptide chip.

New technologies are needed that can diagnose cancer more rapidly and accurately. These technologies must also have the ability to identify the particular cellular abnormalities contributing to the malignancy, thus directing the appropriate treatments. Such technologies should permit absolute quantitation of specific tumor biomarkers and their level of posttranslational modifications. Quantitative molecular profiling of cancer signaling networks would provide a more detailed understanding of the contribution of protein expression and posttranslational modification levels to tumorigenesis. We have developed a unique approach for absolute quantitation of protein expression that integrates affinity capture of proteolytic peptides with mass spectrometry and thus provides detection, identification, and quantitation of their cognate proteins. We have previously shown the high sensitivity and specificity of this approach. Here we demonstrate the absolute quantitation of a model peptide using our technology. We have used this approach to capture epitope-containing peptides from proteolytically digested target proteins, including p53, epidermal growth factor receptor (EGFR), and prostate-specific antigen (PSA). Our technology can easily be extended to the absolute quantitation of protein modification levels, in addition to the determination of protein expression levels, and can be readily adapted for use in a microarray format. This method offers an improved approach to protein chip technology that should prove useful for clinical diagnosis and drug development applications.

Antibodies, Monoclonal↗