Amino acid sequence determination of human S100A12 (P6, calgranulin C, CGRP, CAAF1) by tandem mass spectrometry.

S100A12 has been isolated from human neutrophils. The molecular weight and the amino acid sequence of S100A12 was determined by electrospray-mass spectrometry, tandem mass spectrometry and Edman microsequence analysis. Interestingly, a sequence comparison of S100A12 with all known human S100 proteins revealed that S100A12 is the most divergent of the S100 proteins.

Amino acid sequence analysis of human S100A7 (psoriasin) by tandem mass spectrometry.

The Ca(2+)-binding proteins regulate a number of cellular and extracellular activities and deregulations of S100 gene expression are associated with several human diseases. For example, S100A7 is upregulated in psoriatic skin, implicating a link with psoriasis, a chronic inflammatory dermatosis. We purified human S100A7 and determined its protein sequence by tandem mass spectrometry and Edman microsequence analysis. Interestingly, a sequence comparison of S100A7 with all known human S100 proteins showed that S100A7 is the most divergent of all S100 proteins.

Mass spectrometry and amino acid sequencing of two cadmium-binding metallothionein isoforms from the terrestrial gastropod Arianta arbustorum.

1. Two cadmium-binding metallothionein (Mt) isoforms, called Mta and Mtb, were isolated from terrestrial snails (Arianta arbustorum), using various chromatographic techniques, such as gel-permeation chromatography and reversed-phase HPLC. The purified proteins were S-methylated and cleaved by means of different enzymes (trypsin, endoproteinase Glu-C, and endoproteinase Asp-N). Amino acid sequences were determined by automated Edman degradation and collision-induced dissociation (CID) tandem MS. According to their primary structures, both isoforms should be attributed to class-I Mts. 2. The two forms are structurally identical, differing only by one amino acid exchange in position 60 of the peptide chain. Both isoproteins consist of 66 amino acids, 18 of which are cysteine residues. Most of the cysteine residues are arranged in seven Cys-Xaa-Cys motifs. Mta and Mtb possess an N-terminal acetylated-serine residue and contain a short N-terminal motif which shows a high degree of similarity with the N-termini of histones H4 and H2A. 3. A comparison of Mta and Mtb with other invertebrate Mts shows a very high degree of sequence similarity with a cadmium-binding Mt from Helix pomatia, a species that is closely related to Arianta arbustorum. Moreover, Mta and Mtb, as expected, also exhibit structural similarities with Mts from other molluscan species, such as mussels and oysters. It is suggested that Mta and Mtb represent two allelic isoforms, reflecting the genetic polymorphism of Mt in Arianta arbustorum.

Isolation, primary structures and metal binding properties of neuronal growth inhibitory factor (GIF) from bovine and equine brain.

Human neuronal growth inhibitory factor (GIF) impairs the survival of cultured neurons and is deficient in the brains of Alzheimer's disease victims. We have isolated and sequenced analogous proteins from bovine and equine brain. By comparing their primary structures with those of human, mouse and rat GIFs, a consensus GIF sequence was obtained. Although this exhibits ca. 65% similarity with primary structures of mammalian metallothioneins (MTs), some significant differences are expected in the content of helix and turn secondary structures. In contrast to MTs, which usually bind 7 Zn(II) ions, human, bovine and equine GIFs contain 1-4 Cu(I) and 3-5 Zn(II) ions in species-specific ratios. The observed Cu(I) phosphorescence (lambda max, 550-590 nm; tau, 100 microseconds at 77 K) indicates the presence of the cuprous ion. Both bovine Cu1Cd5- and the equine Cu3Cd3-GIF derivatives (Cd replacing Zn) exhibit cadmium-dependent absorption and CD features between 220-260 nm characteristic of Cd-thiolate clusters similar to those in Cd-MTs.

Purification and primary structure of snail metallothionein. Similarity of the N-terminal sequence with histones H4 and H2A.

A cadmium-binding metallothionein has been purified from metal-exposed Roman snails (Helix pomatia) using gel-permeation, ion-exchange and reverse-phase high-performance liquid chromatography. The S-methylated protein was digested with trypsin and the endoproteinases Asp-N, Glu-C and Arg-C. While most of the resulting peptides could be sequenced by Edman degradation, the intact protein, as well as the N-terminal peptide, proved to be blocked. Analysis by mass spectrometry showed that the N-terminal amino acid was an acetylated serine residue. Snail metallothionein, which is suggested to be involved in the detoxification of cadmium, contains 66 amino acid residues, 18 of which are cysteine residues arranged in seven Cys-Xaa-Cys motifs. The calculated molecular mass of the protein is 6.62 kDa. The primary structure of snail metallothionein reveals a clear relationship with molluscan and vertebrate metallothioneins, but lower similarity with metallothioneins of other invertebrate species. The N-terminal region of the isolated protein proved to be unique among the metallothionein sequences determined so far, showing high degrees of similarity with the N-terminal sequences of histones H2A and H4 which may be important for regulatory functions.

The polyelectrolyte properties of elastin.

The charge structure and ionic interactions of elastin prepared from the pig thoracic aorta by acid, alkali, or CNBr extraction have been investigated by potentiometric titration and radiotracer techniques. The number of charged groups was consistent with the amino acid composition, comparable to elastin from other sources and insensitive to the method of preparation. The enthalpies of ionization of the basic groups were comparable for those previously found for proteins but those of the acidic groups were higher. Ionic interactions were predominantly electrostatic although a strong affinity for chloride ions was noted. Changes in ionic interactions as the elastin was stretched had a similar effect to an increase in the apparent fixed charge density of the tissue. Mechanical strain altered the protonation of the elastin and the pK of the carboxyl groups. Conversely, the conformation of the elastin network varied with ionic strength and pH, being particularly sensitive to the degree of ionization of the more basic groups and with the ionic strength and anion composition of the medium. We speculate that strain induced changes in the conformation of elastin altering its reactivity towards lipids, ions or matrix macromolecules or changes in its mechanical properties resulting from changes in its ionic environment may be of physiological or pathological importance.