Search

After tryptic digestion of protein Z a peptide corresponding to residues 1-43 has been isolated and sequenced. Smaller peptides were generated by treatment of the peptide with pepsin and then separated by high-performance liquid chromatography. The purity of the peptides was sufficient for mass spectrometric measurement. Thirteen gamma-carboxyglutamic acid residues have been identified and the peptide shows strong homology to the corresponding domains of the classical vitamin-K-dependent blood clotting proteins.

The domain organization and the post-translational modifications of human placenta calreticulin were analysed by MS in combination with proteolytic digestion. Prolonged treatment with trypsin, chymotrypsin, elastase, Staphylococcus aureus V8 protease, or proteinase K all led to a 6- to 7-kDa decrease in the molecular mass of calreticulin. The decrease was found to be due to cleavages in the region around residue 340. In addition, minor fragments resulting from secondary cleavages close to the N-terminus were observed, but no stable fragments of intermediate size were found. These results show that the C-domain of calreticulin is susceptible to proteolytic cleavage and that the N- and P-domains form a proteolytically stable tight association. A disulfide bridge between the first two cysteines was mapped in the N-domain, and the third cysteine was found in the reduced form. No post-translational modifications in the form of glycosylation or phosphorylation were found. A modified form of calreticulin lacking the C-terminal hexapeptide including the KDEL endoplasmic reticulum retention sequon was isolated. Such a truncation may point to a mechanism that allows escape of calreticulin from the endoplasmic reticulum.

We have used matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for mass determination of pigments and proteins in chlorosomes, the light-harvesting organelles from the photosynthetic green sulfur bacterium Chlorobium tepidum. By applying a small volume (1 µL) of a concentrated suspension of isolated chlorosomes directly to the target of the mass spectrometer we have been able to detect bacteriochlorophyll a and all the major homologs of bacteriochlorophyll c. The peak heights of the different bacteriochlorophyll c homologs in the MALDI spectra were proportional to peak areas obtained from HPLC analysis of the same sample. The same result was also obtained when whole cells of Chl. tepidum were applied to the target, indicating that MALDI-MS can provide a rapid method for obtaining a semiquantitative determination or finger-print of the bacteriochlorophyll homologs in a small amount of green bacterial cells. In addition to information on pigments, the MALDI spectra also contained peaks from chlorosome proteins. Thus we have been able with high precision to confirm the molecular masses of the chlorosome proteins CsmA and CsmE which have been previously determined by conventional biochemical and genetic methods, and demonstrate the presence of truncated versions of CsmA and CsmB.

Interferon-gamma (IFN-gamma) is a secretory glycoprotein produced by T cells in response to antigenic or mitogenic stimuli. We studied the kinetics of the synthesis, N-glycosylation, and secretion of IFN-gamma in human CD8+ T lymphocytes stimulated via T-cell receptor. Highly elevated IFN-gamma mRNA levels were found as early as 1 h after stimulation. Maximal IFN-gamma protein synthesis was observed 2-4 h after induction and appeared to correlate to steady-state IFN-gamma mRNA levels. As analyzed by pulse/chase experiments, the secretion of IFN-gamma from T cells was very rapid, the secretion half-time being approximately 20-25 min. Inhibition of N-glycosylation by tunicamycin dramatically reduced the expression of IFN-gamma, but did not block its secretion. Natural IFN-gamma is heterogeneously glycosylated and doubly, singly, and unglycosylated forms exist. Experiments performed in a cell-free translation/glycosylation system with mutated IFN-gamma constructs lacking either one of the potential glycosylation sites suggested that Asn25 is more efficiently glycosylated than Asn97. Site-specific oligosaccharide analysis of natural IFN-gamma by glycosidase treatment followed by matrix-assisted-laser-desorption-ionization mass spectrometry revealed considerable variation in the carbohydrate structures, with more than 30 different forms. The glycans at Asn25 consisted of fucosylated, mainly complex-type oligosaccharides, whereas the glycans at Asn97 were more heterogeneous, with hybrid and high-mannose structures. Our results emphasize the essential role of N-linked glycans in the biology of IFN-gamma and show that there is a considerable heterogeneity in the individual sugar chains of this important human cytokine.

Amyloid P component (AP) is a naturally occurring glycoprotein that is found in serum and basement membranes. AP is also a component of all types of amyloid, including that found in individuals who suffer from Alzheimer's disease and Down's syndrome. Because AP has been found to bind strongly and specifically to certain glycosaminoglycans that are components of amyloid deposits, AP may play an important role in the maintenance of amyloid. In the present work, we isolated and identified two proteolytic fragments of AP that are responsible for its heparin-binding activity. Neither fragment corresponds to published heparin-binding sequences. The structural requirements for activity of the peptides (amino acid residues 27-38 and 192-203 of AP) were examined by means of solid-phase inhibition assays with synthetic peptides. AP-(192-203)-peptide inhibits the Ca(2+)-dependent binding of AP to heparin with an IC50 of 25 microM, while the IC50 of AP-(27-38)-peptide and AP-(33-38)-peptide are 10 microM and 2 microM, respectively. The understanding of the structure and function of active AP peptides will be useful for development of amyloid-targeted diagnostics and therapeutics.

We have elucidated the complete amino acid sequence of one of the avenin components, avenin-3, isolated from oat (Avena sativa L.), variety Narymsky 943. The sequence of the protein was determined by sequencing of CNBr and trypsin-generated peptides in combination with mass spectrometry. The protein is a single polypeptide chain, consisting of 201 amino acid residues with M(r) 23,252.8. The N-terminal amino acid residue of the protein is blocked with 5-oxoproline (pyroglutamic acid). All eight cysteine residues in avenin-3 are involved in disulphide bonds. The positions of these bonds were established by identification of a CNBr cleavage product of the intact avenin containing all the disulfide bonds (S-S core). Subsequent subdigestion of this S-S core allowed isolation of disulphide bonded peptides detected by differential reverse-phase HPLC before and after reduction. As a result, all four disulphides Cys50 Cys183, Cys58 Cys77, Cys84 Cys85 and Cys97 Cys191 were identified. Comparison of avenins with other prolamins demonstrates a high degree of similarity, which is especially pronounced around the cysteine residues. Avenins differ slightly from other prolamins in having unique N-terminal sequences and some differences in the repeated sequence motifs.

The primary structures of two basic low-molecular-mass proteins, Lm-67 and Lm-70 from the pharate cuticle of the migratory locust, Locusta migratoria, were determined. The sequencing strategy was based on combined use of plasma--desorption mass spectrometry (PDMS) and automatic Edman degradation of the proteins and their enzymically derived peptides. The mass-spectral data showed the presence of two proteins in each preparation. For protein preparation Lm-67, this was indicated by the mass spectrum of the intact protein. For protein preparation Lm-70, the presence of two variants only became evident by mass-spectrometric analysis of the enzymically derived peptides. Both proteins show strong similarity to other exocuticular proteins from L. migratoria.

The complete structure of protein isolated from endocuticle of sexually mature locusts, Locusta migratoria, has been determined by a combination of automatic Edman degradation and plasma desorption mass spectrometry. The protein is extensively post-translationally modified. The N-terminal is 5-oxoproline (pyroglutamic acid) and the C-terminal proline residue is amidated. Furthermore, the protein is glycosylated by a single N-acetyl-galactosamine residue at one, two or three threonines. The N-terminal sequence was obtained by analysing the N-acetylated N,O-permethylated derivative using plasma desorption mass spectrometry. The position and type of carbohydrate were determined by combining an HPLC-based carbohydrate analysis with the peak pattern of the phenylthiohydantoin derivative in automatic sequencing and with mass information on peptides. The protein has pronounced similarity to cuticular proteins from larvae of diptera and lepidoptera, but only slight resemblance to the previously sequenced locust exocuticular proteins. This indicates a similarity between soft larval cuticles and locust endocuticle, a similarity which may extend to their mechanical properties.

We have now demonstrated that activated complement component C1s cleaves beta 2-microglobulin at the position identical to that at which beta 2-microglobulin is cleaved in serum of patients suffering from lung cancer. The main cleavage is in the disulphide loop C-terminal to Lys-58, generating a modified form of beta 2-microglobulin with a two-chain structure. The C-terminal Lys-58 in the A chain is highly susceptible to removal by a carboxypeptidase-B-like activity causing the formation of des-Lys58-beta 2-microglobulin. This is the first demonstration of a noncomplement protein substrate for the proteolytic activity of C1s. The C1s-induced cleavage of beta 2-microglobulin can be inhibited in the presence of C1 esterase inhibitor, demonstrating a regulatory function of C1 esterase inhibitor in the C1s-induced cleavage of beta 2-microglobulin.