A new low-molecular-mass (6767.8 Da) serine proteinase isoinhibitor has been isolated from oil-rape (Brassica napus var. oleifera) seed, designated 5-oxoPro1-Gly62-RTI-III. The 5-oxoPro1-Gly62-RTI-III isoinhibitor is longer than the Asp2-Pro61-RTI-III and the Ser3-Pro61-RTI-III forms, all the other amino acid residues being identical. In RTI-III isoinhibitors, the P1-P1' reactive site bond (where residues forming the reactive site have been identified as PnellipsisP1 and P1'ellipsisPn', where P1-P1' is the inhibitor scissile bond) has been identified at position Arg21-Ile22. The inhibitor disulphide bridges pattern has been determined as Cys5-Cys27, Cys18-Cys31, Cys42-Cys52 and Cys54-Cys57. The disulphide bridge arrangement observed in the RTI-III isoinhibitors is reminiscent of that found in a number of toxins (e.g. erabutoxin b). Moreover, the organization of the three disulphide bridges subset Cys5-Cys27, Cys18-Cys31 and Cys42-Cys52 is reminiscent of that found in epidermal growth factor domains. Preliminary 1H-NMR data indicates the presence of alphaalphaNOEs and 3JalphaNH coupling constants, typical of the beta-structure(s). These data suggest that the three-dimensional structure of the RTI-III isoinhibitors may be reminiscent of that of toxins and epidermal growth factor domains, consisting of three-finger shaped loops extending from the crossover region. Values of the apparent association equilibrium constant for RTI-III isoinhibitors binding to bovine beta-trypsin and bovine alpha-chymotrypsin are 3.3 x 109 m-1 and 2.4 x 106 m-1, respectively, at pH 8.0 and 21.0 degrees C. The serine proteinase : inhibitor complex formation is a pH-dependent entropy-driven process. RTI-III isoinhibitors do not show any similarity to other serine proteinase inhibitors except the low molecular mass white mustard trypsin isoinhibitor, isolated from Sinapis alba L. seed (MTI-2). Therefore, RTI-III and MTI-2 isoinhibitors could be members of a new class of plant serine proteinase inhibitors.
This paper reports the purification and localization of a Tuber borchii Vittad, fruitbody protein (TBF-1) and the cloning of the encoding gene. TBF-1 is detectable by SDS-PAGE analyses only in this white truffle species and presents a molecular mass of 11,994 Da. TBF-1 was purified by one-step Reversed-Phase HPLC and its complete amino acid sequence was determined after digestion with trypsin and N-Asp endoproteinase. Polyclonal antibodies were produced and tested in immunofluorescence and immunogold experiments, providing information about the protein localization. It was detected mostly on the hyphal walls, where it was colocalized with beta-1,3-glucans and chitin. The sporal wall was not labeled. The encoding gene (tbf-1) was cloned using several techniques involving PCR. The coding region consists of a 360-bp open reading frame interrupted by an intron, with another intron following the stop codon. A putative signal peptide of 12 amino acids was found at the N-terminal. Northern blot analysis revealed that tbf-1 is highly expressed in unripe and ripe fruitbodies and was not detectable in culture mycelium or ectomycorrhizal roots.
MEN 11300 is a hybrid glycoprotein of 297 amino acids obtained by fusion of the cDNA encoding GM-CSF with the cDNA encoding EPO followed by transfection of the hybrid gene into CHO cells. The oligonucleotide construct incorporated a spacing sequence between the two individual cDNAs which encodes eight amino acids constituting a linker peptide intended to separate the GM-CSF and EPO moieties. The recombinant MEN 11300 protein was submitted to a detailed structural characterization including the verification of the entire amino acid sequence, the assignment of the disulfide bridges pattern, the identification of the glycosylation sites and the definition of the glycosidic moiety, including site-specificity. Partial processing of the C-terminal Arg residue and the occurrence of N-glycosylation sites at Asn27, Asn155, Asn169, Asn214 were established. Moreover, O-glycosylation at Ser257 and at the N-terminal region was also detected. A large heterogeneity was observed in the N-glycans due to the presence of differently sialylated and fucosylated branched complex type oligosaccharides whereas O-linked glycans were constituted by GalGalNAc chains with a different number of sialic acids. The disulfide bridges pattern was established by direct FABMS analysis of the proteolytic digests or by ESMS analysis of HPLC purified fractions. Pairing of the eight cysteine residues resulted in Cys54-Cys96, Cys88-Cys121, Cys138-Cys292, and Cys160-Cys164. This S-S bridges pattern is identical to that occurring in the individual natural GM-CSF and EPO, thus showing that the two protein moieties in MEN 11300 can independently acquire their native three-dimensional structure.
A new strategy for the structural characterisation of human albumin variants has been developed which makes extensive use of mass spectrometric methodologies. The rationale behind the method is to provide a rapid and effective screening of the entire albumin structure. The first step in this strategy consists in the attempt to determine the accurate molecular mass of the intact variant by electrospray mass spectrometry often providing a first indication on the presence of the variant. An HPLC procedure has been developed io isolate all the seven fragments generated by CNBr hydrolysis of HSA in a single chromatographic step. A rapid screening of the entire albumin structure is achieved by the ESMS analysis of the peptide fragments and the protein region(s) carrying the structural abnormality is identified by its anomalous mass value(s). Mass mapping of the corresponding CNBr peptide, either by Fast Atom Bombardment Mass Spectrometry (FABMS) or by Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDIMS), leads to the definition of the site and the nature of the variation. This combined strategy was applied to the structural characterisation of three HSA genetic variants and provided to be an effective procedure for the rapid assessment of their structural modifications showing considerable advantages over the classical approach.
A long-term electrophoretic survey on plasma proteins, which was carried out in several clinical laboratories in Italy, identified 28 different genetic variants of human serum albumin and four cases of analbuminemia. We have previously characterized 16 point mutations, 3 C-terminal mutants, and the genetic defects in two analbuminemic subjects. Here, we report the molecular defects of four alloalbumins that have been characterized by protein structural analysis. Of these, three represent new single-point mutations: albumins Tregasio, Val122-->Glu, Bergamo, Asp314-->Gly, and Maddaloni, Val533-->Met. The fourth, albumin Besana Brianza, has the same Asp494-->Asn mutation that introduces a glycosylation site which has been previously reported in a variant from New Zealand, albumin Casebrook. However, in contrast to albumin Casebrook, albumin Besana Brianza is only partially glycosylated and the oligosaccharide is heterogeneous, consisting of a biantennary complex type N-glycan with either two or one sialic acid residue(s) on the antennae. Both albumin Maddaloni and Besana Brianza represent mutations at hypermutable CpG dinucleotide sites; albumin Maddaloni is a mutant that does not involve a charged amino acid.
The alpha and beta chains from human recombinant gonadotropins follitropin, lutropin and choriogonadotropin expressed in CHO cells have been structurally characterised both at the protein and at the carbohydrate level by using advanced mass spectrometric procedures. The three alpha chains share the same amino acid sequence while they display different glycosylation patterns. The oligosaccharide structures detected belong to the complex-type glycans with different degree of sialylation. Partial proteolytic processing occurred at the N-terminus of the follitropin beta chain and at the C-terminus of the lutropin beta chain. The N-linked glycans from the three beta chains were found to contain fucosylated and sialylated diantennary and triantennary complex-type structures. The follitropin beta chain showed the presence of N-acetyllactosamine repeats on the antennae. The overall structure of the recombinant glycohormones corresponds to their natural counterparts with the exception that sulphated terminal glycosylation is missing.
Structural and biochemical characteristics of transglutaminase purified by a rapid chromatographic procedure from the rat coagulating gland (anterior prostate) secretion are reported. Fast atom bombardment mapping and automated Edman degradation experiments allowed us to verify that at least 85% of the entire transglutaminase amino acid sequence is identical to that derived from the cDNA of the major androgen-dependent rat prostate protein called DP1. The enzyme was found NH2 terminally blocked and largely post-translationally modified, since the presence of N-linked oligosaccharides, as well as of complex lipidic structures, was observed. Mass spectral analysis showed that Asn-408 and -488 are the glycosylated sites, the N-linked structures identified belonging to both high-mannose and complex type glycans. The presence of myo-inositol, of glycerol bound fatty acids, and the high content of mannose residues, are in agreement with previous observations suggesting that a lipid anchor is bound to coagulating gland secretion transglutaminase. Furthermore, two tightly bound calcium ions per molecule of enzyme were detected. Finally, a strong stimulation of the enzyme activity in vitro by both SDS and a variety of phosphatidic acids was observed. The reported structural and functional peculiarities should definitively lead to consider the prostate enzyme as a new member (type IV) of the transglutaminase family.
A member of the laccase multigene family in Pleurotus ostreatus has been cloned and sequenced. The gene structure has been determined by comparison with the corresponding cDNA, synthesized by reverse transcription/PCR amplification. The gene encode a laccase isoenzyme of 533 amino acids which has already been purified and characterized [Palmieri, G., Giardina, P., Marzullo, L., Desiderio, B., Nitti, G., Cannio, R. & Sannia, G.(1993) Appl. Microbiol. Biotechnol. 39, 632-636]. More than 92% of the protein sequence, including the N and C termini, has been verified by fast-atom-bombardment mass spectrometry, thus confirming the correspondence between the gene and its protein product. The protein was N-glycosylated Asn444. Glycan analysis showed the presence of only a high-mannose structure containing varying numbers of mannose residues. The presence of O-linked oligosaccharides as well as other post-translational modification could be ruled out by the mass analysis.
Saposins A, B, C, and D are a group of homologous glycoproteins derived from a single precursor, prosaposin, and apparently involved in the stimulation of the enzymatic degradation of sphingolipids in lysosomes. All saposins have six cysteine residues at similar positions. In the present study we have investigated the disulfide structure of saposins B and C using advanced mass spectrometric procedures. Electrospray analysis showed that deglycosylated saposins B and C are mainly present as 79- and 80-residue monomeric polypeptides, respectively. Fast atom bombardment mass analysis of peptide mixtures obtained by a combination of chemical and enzymatic cleavages demonstrated that the pairings of the three disulfide bridges present in each saposin are Cys4-Cys77, Cys7-Cys71, Cys36-Cys47 for saposin B and Cys5-Cys78, Cys8-Cys72, Cys36-Cys47 for saposin C. We have recently shown that saposin C interacts with phosphatidylserine-containing vesicles inducing destabilization of the lipid surface (Vaccaro, A. M., Tatti, M., Ciaffoni, F., Salvioli, R., Serafino, A., and Barca, A. (1994) FEBS Lett. 349, 181-186); this perturbation promotes the binding of the lysosomal enzyme glucosylceramidase to the vesicles and the reconstitution of its activity. It was presently found that the effects of saposin C on phosphatidylserine liposomes and on glucosylceramidase activity are markedly reduced when the three disulfide bonds are irreversibly disrupted. These results stress the importance of the disulfide structure for the functional properties of the saposin.