Solution structure of the amino acid sequence coded by the rarely expressed exon 26A of human elastin: the N-terminal region.

We previously reported the structural and biological properties of the C-terminal sequence (REGDPSSSQHLPSTPSSPRV) coded by the rarely expressed exon 26A of human elastin. It assumes a stable type II beta-turn structure spanning the REGD sequence and possesses chemotactic and immunological properties. Here the structural characterization of the sequence coded by this exon was completed. Nuclear magnetic resonance and circular dichroism studies on the N-terminal amino acid sequence (GADEGVRRSLSPELREGD) showed the presence of an alpha-helix within VRRSL and a type II beta-turn within SPEL. The smaller peptides GADEGVRRSLSP and LSPELREGD revealed structural features similar to those identified in the parent peptide. No beta-turn was found in the REGD sequence of these peptides and no chemotactic activity was detected, thereby demonstrating that this biological activity is conformation dependent. Structural studies on additional peptides such as LREGD, ELREGD and LSPELREGDPSS showed that the presence of a Glu residue two positions before the Arg residue inhibits the beta-turn formation in the REGD sequence.     

Molecular and supramolecular structural studies on human tropoelastin sequences

One of the unusual properties of elastin is its ability to coacervate, which has been proposed to play an important role in the alignment of monomeric elastin for cross-linking into the polymeric elastin matrix. The temperature at which this transition takes place depends on several factors including protein concentration, ionic strength, and pH. Previously, polypeptide sequences encoded by different exons of the human tropoelastin gene have been analyzed for their ability to coacervate and to self-assemble. Few of them were indeed able to coacervate and only one, that encoded by exon 30 (EX30), gave amyloid fibers. In this article, we report on two chemically synthesized peptides-a decapeptide and an octadecapeptide-whose sequences are contained in the longer EX30 peptide and on a polypeptide (EX1-7) of 125 amino-acid residues corresponding to the sequence coded by the exons 1-7 and on a polypeptide (EX2-7) of 99 amino-acid residues encoded by exons 2-7 of human tropoelastin obtained by recombinant DNA techniques. Molecular and supramolecular structural characterization of these peptides showed that a minimum sequence of approximately 20 amino acids is needed to form amyloid fibers in the exon 30-derived peptides. The N-terminal region of mature tropoelastin (EX2-7) gives rise to a coacervate and forms elastinlike fibers, whereas the polypeptide sequence containing the signal peptide (EX1-7) forms mainly amyloid fibers. Circular dichroism spectra show that beta-structure is ubiquitous in all the sequences studied, suggesting that the presence of a beta-structure is a necessary, although not sufficient, requirement for the appearance of amyloid fibers.     

Molecular mobility of elastin: effect of molecular architecture

The thermal and dielectric properties of elastin and two soluble derivatives (kappa-elastin and derived elastin peptides from enzymatic elastolysis) were investigated in the freeze-dried state in a wide temperature range (from -180 to +220 degrees C). The glass transition of these amorphous proteins was studied by differential scanning calorimetry (DSC). The dielectric relaxations of both proteins were followed by thermally stimulated currents (TSC), an isochronal dielectric spectrometry running at variable temperature, analogous to a low-frequency spectroscopy (10(-3)-10(-2) Hz) and by dynamic dielectric spectroscopy (DDS), performed isothermally with the frequency varying from 10(-2) to 3 x 10(6) Hz. The combination of TSC and DDS experiments and the determination of the activation parameters of the relaxation times inform about the molecular mobility of the proteins, both in the glassy state and in the liquid state. Major differences between the relaxation behavior of elastin and its soluble derivatives have been discussed and correlated with the molecular architecture of the proteins.     

Domains in tropoelastin that mediate elastin deposition in vitro and in vivo

Elastic fiber assembly is a complicated process involving multiple different proteins and enzyme activities. However, the specific protein-protein interactions that facilitate elastin polymerization have not been defined. To identify domains in the tropoelastin molecule important for the assembly process, we utilized an in vitro assembly model to map sequences within tropoelastin that facilitate its association with fibrillin-containing microfibrils in the extracellular matrix. Our results show that an essential assembly domain is located in the C-terminal region of the molecule, encoded by exons 29-36. Fine mapping studies using an exon deletion strategy and synthetic peptides identified the hydrophobic sequence in exon 30 as a major functional element in this region and suggested that the assembly process is driven by the propensity of this sequence to form beta-sheet structure. Tropoelastin molecules lacking the C-terminal assembly domain expressed as transgenes in mice did not assemble nor did they interfere with assembly of full-length normal mouse elastin. In addition to providing important information about elastin assembly in general, the results of this study suggest how removal or alteration of the C terminus through stop or frameshift mutations might contribute to the elastin-related diseases supravalvular aortic stenosis and cutis laxa.     

Human leukocyte elastase hydrolysis of peptides derived from human elastin exon 24

In normal and pathological tissues, polymorphonuclear leukocyte proteases (elastase, cathepsin G and proteinase 3) may generate soluble peptides through limited proteolysis of elastin, the main component of mature elastic fibres. Elastin-derived peptides display diverse biological activities including cell migration, differentiation, proliferation, chemotaxis, tumor progression and up-regulation of metalloproteinases. To be biologically active, their structures must adopt a beta-turn conformation which accommodates to the cell surface-located elastin binding protein. In this study, we established that human elastin exon 24-derived peptides are hydrolysed by leukocyte elastase, when the active site is fully occupied (from S(5) to S'(3)). As shown by mass spectrometry analyses, a major cleavage site was demonstrated at a Val-Ala bond and a minor one at Gly-Val bond. For longer peptides, the hydrolysed fragments could themselves be re-hydrolysed. If the shortest fragments do not contain the GxxPG sequence known to stimulate cellular effects, some of the intermediates together with hydrolysis fragments generated by other proteases such as proteinase 3, may possess this motif.     

A turning point in the knowledge of the structure-function-activity relations of elastin]

In this review are presented the last new results of our research group dealing with the molecular structures (atomic level) of tropoelastin, elastin and elastin derived peptides studied by using essentially methods of bioinformatics (theoretical predictions and molecular modelling) linked to experimental circular dichroism spectroscopic studies. We already had characterized both the local secondary structure and some parts of the tertiary structure of the tropoelastin and elastin molecules (human, bovine...), by using either theoretical predictions (local secondary structure, linear epitopes...) and/or experimental data (optical spectroscopic methods: Raman scattering, infrared absorption, circular dichroism). Except the cross-linking regions which are in helical conformations, the whole tropoelastin structure displays a lot of beta-reverse turns which usually belong to irregular structures in proteins. These turns play a key role in other regularly structures orientation (alpha-helix, beta-strand), thus they are very important in the native protein 3D architecture. It is particularly true for human tropoelastin, because its sequence is rich in glycines and prolines, and these residues are frequently met in beta-turns (a beta-turn is made of four consecutive residues which are stabilized by an hydrogen bond). Several types of beta-turns can be defined with the dihedral angles values phi and psi of the two central residues. Thus, by using a very recent updated set of propensities for the amino acid residues to belong to given types of reverse beta-turns (extracted from a reference set of known 3-D structures of globular proteins), we have determined, (by using our home made software COUDES), for all possible tetrapeptides of the human tropoelastin sequence, the distribution and the characterization of the possible type of turns. Thus, it is shown that the locations and/or the types of these reverse beta-turns reveal a regularity and are not all random. This confirms our hypothesis that intra-molecular elasticity of tropoelastin could be explained by the possibility of transitions between conformations involving short beta-strands and beta-turns. This result is of great interest in the construction (by using molecular biology) of elastic biomaterials derived from the elastin sequence (particularly, the elastin derived peptides corresponding to the sequence exon 21--(exon 24--exon 24...). Our study permit also to predict the conformations of specific elastin derived peptides which could have interesting biological activity. Peptides resulting from the degradation of elastin, the insoluble polymer of tropoelastin and responsible for the elasticity of vertebrate tissues, can induce biological effects and notably the regulation of matrix metalloproteinases (MMP-s) activity. Recently, it was proposed that some elastin derived hexapeptides resulting from circular permutations of VGVAPG (a three fold repetition sequence in exon 24 of human tropoelastin) possess MMP-1 production and activation regulation properties. This effect depends on the presence of the tropoelastin specific membraneous receptor 67 KDa EBP (Elastin Binding Protein). Our results obtained by using both circular dichroism spectroscopy and linear predictions confirmed the hypothesis of a structure dependent mechanism with a possibly occurring type VIII beta-turn on the first four residues of the GXXPG sequence consensus which is only present among all active peptides. Thus, we have performed extensive molecular dynamics studies, in both implicit and explicit solvent, on these active and inactive elastin derived hexapeptides. Using our own analysis method of pattern recognition of the types of the beta-reverse-turns followed during the molecular dynamics trajectory, we found that active and inactive peptides effectively form two well distinct conformational groups in which active peptides preferentially adopt conformation close to type VIII GXXP (beta-reverse-turn. The structural role of the C terminal G residue could also be explained. Additional molecular simulations on (VGVAPG)2 and (VGVAPG)3 show the formation of two or three GXXP tetrapeptides adopting a structure close to type VIII beta-reverse-turn, suggesting a local conformational preference for this motif. This observation of a specific structural single and/or repeated motif is in agreement with the circular dichroism spectra of the involved (VGVAPG)1, (VGVAPG)2 and (VGVAPG)3 peptides and then it can be proposed that their biological activities have to be linear. The final aim of this type of work is to understand more about the sequence/structure/function/activity relationships of those structured peptides in order to propose specific sequences (corresponding to specific structures) for best biological activity results.     

Characterization of peptides resulting from digestion of human skin elastin with elastase

Several pathological disorders are associated with abnormalities in elastic fibers, which are mainly composed of elastin. Understanding the biochemical basis of such disorders requires information about the primary structure of elastin. Since the acquisition of structural information for elastin is hampered by its extreme insolubility in water or any organic solvent, in this study, human skin elastin was digested with elastase to produce water-soluble peptides. Tandem mass spectrometry (MS/MS) experiments were performed using conventional electrospray ionization (ESI) and nano-ESI techniques coupled with ion trap and quadrupole time-of-flight (qTOF) mass analyzers, respectively. The peptides were identified from the fragment spectra using database searching and/or de novo sequencing. The cleavage sites of the enzyme and, for the first time, the extent and location of proline hydroxylation in human skin elastin were determined. A total of 117 peptides were identified with sequence coverage of 58.8%. It has been observed that 25% of proline residues in the sequenced region are hydroxylated. Elastase cleaves predominantly at the C-terminals of the amino acids Gly, Val, Leu, Ala, and Ile, and to a lesser extent at Phe, Pro, Glu, and Arg. Our results confirm a previous report that human skin elastin lacks amino acid sequences expressed by exon 26A.     

Elements of the rat tropoelastin gene associated with alternative splicing

Multiple isoforms of tropoelastin, the soluble precursor of elastin, are the products of translation of splice-variant mRNAs derived from the single-copy tropoelastin gene. Previous data had demonstrated DNA sequence heterogeneity in three domains of rat tropoelastin mRNA, indicating alternative splicing of several exons of the rat tropoelastin gene. Rat tropoelastin genomic clones encompassing the sites of alternative splicing were isolated and sequenced. Two sites of alternative splicing identified in rat tropoelastin mRNA sequences corresponded to exons 13-15 and exon 33 of the rat tropoelastin gene. Furthermore, the variable inclusion of an alanine codon in exon 16 resulted from two functional acceptor sites separated by three nucleotides. DNA sequences flanking exons subject to alternative splicing were analyzed. These exons contained splicing signals that differed from consensus sequences and from splicing signals of constitutively spliced exons. Introns immediately 5' of exons 14 and 33, for example, lacked typical polypyrimidine tracts and had weak, overlapping branch point sequences. Further, a region of secondary structure encompassing the acceptor site of exon 13 may influence alternative splicing of this exon. These results demonstrate that multiple cis-acting sequence elements may contribute to alternative splicing of rat tropoelastin pre-mRNA.     

Proteinase 3 hydrolysis of peptides derived from human elastin exon 24

Summary. In normal and pathological tissues, elastin-derived peptides proceed of elastin degradation by polymorphonuclear leukocyte proteases: elastase, cathepsin G and proteinase 3. They were demonstrated to have a chemotactic activity, to promote cell proliferation and protease release, . . .. To be biologically active, their structures, which reflect elastase specificity, must adopt a β-turn conformation which accommodate to the cell surface-located elastin binding protein. In this study, we establish that human elastin exon 24-derived peptides containing at least two repeated VGVAPG sequences are hydrolyzed by the proteinase 3 (Pr3). As shown by mass spectrometry analyses, the demonstrated cleavage sites are in agreement with previously reported Pr3 substrate specificity and its lengthy substrate binding site. The characterization of the Pr3-generated products indicate that they contain at least one GXXPG sequence known to stimulate cellular effects after binding to the elastin receptor.     

Rat atrial natriuretic factor: Isolation,structure and biological activities of four major peptides

Four peptides possessing both natriuretic activity and smooth muscle relaxant activity were isolated from rat atrium and their amino acid sequences determined. The four peptides designated ANF-I, ANF-II, ANF-III and ANF-IV containing 35, 31, 30 and 25 amino acid residues, respectively, were obtained in a molar ratio of 4:60:20:16. The predominant species ANF-II, which may represent the native form of ANF, has the following sequence: (H₂N)-G-P-R-S-L-R-R-S-S-C-F-G-G-R-I-D-R-I-G-A-Q-S-G-L-G-C-N-S-F-R-Y-(COOH) in which Cys-10 and Cys-26 are disulfide linked. Cleavage of the aspartyl linkage at position 16 by staphylococcal protease caused complete inactivation, indicating that the ring conformation is essential. The dose-response relationships determined for the four pepdides in relaxing norepinephrine-induced contraction of rabbit thoracic aorta showed half-maximum relaxation at concentrations ranging from 1.5 × 10^?9 to 2.5 × 10^?9 M. Comparable dose-response relationships were observed in relaxation of carbacol-induced contraction of chick rectum strips as tested with ANF-II and ANF-IV.     

Mechanisms of interaction between human skin fibroblasts and elastin: differences between elastin fibres and derived peptides.

3H-Labelled kappa-elastin peptides (kE:75 kDa molecular weight) were shown to bind to confluent human skin fibroblast (HSF) cultures in a time-dependent and saturable manner. Scatchard analysis indicated the presence of high affinity binding sites with kD = 2.7 x 10(-10) M and 19,000 sites per cell. Binding of kE to its receptor on HSF accelerates and intensifies the adhesion of insoluble elastin fibres (iE) to confluent HSF. Optimal effect was attained for a kE concentration of 0.3 x 10(-9) M close to kD. This stimulatory effect of kE on the binding of iE to HSF could be inhibited by neomycin, retinal and pertussis toxin, substances which act at different levels of the transduction mechanism following the activation of the receptor and the subsequent triggering of cell biological events (chemotaxis, modification of calcium fluxes). The stimulation of iE adhesion to HSF induced by kE as well as kE binding to the cells could be inhibited by lactose and laminin but not by Arg-Gly-Asp-Ser(RGDS) peptides. This indicates that the elastin peptide receptor on HSF possesses lectin-like properties and shares homology with the laminin receptor as also shown for other cell types. None of the substances tested, that is inhibitors of the transduction mechanism, lactose, laminin and Arg-Gly-Asp-Ser(RGDS) peptides were shown to interfere significantly with the binding of iE (in the absence of added kE) to confluent HSF. The proteins adhering strongly to elastin fibres were isolated by a sequential extraction procedure and the final hydrochloride guanidinium-DTT extract was analysed by SDS-PAGE under reducing conditions, Western blots using specific antibodies against several connective tissue proteins and affinity for [3H]-kE following nitrocellulose electro-transfer of proteins. Fibronectin, vitronectin, tropoelastin(s), and a 120 kDa cysteine rich glycoprotein previously designated as elastonectin were identified. Among these proteins, [3H]-kE was found to bind exclusively to a 65 kDa protein that could be eluted selectively from elastin fibres with a neutral buffer containing 100 mM lactose. Therefore the elastin peptide receptor on human skin fibroblasts shares properties with the elastin receptor characterized from other cell types. Conformational differences between elastin peptides and elastin fibres could explain the differences in the mechanisms of interactions between elastin fibres and elastin peptides with HSF in culture. The stimulatory effect of elastin-derived peptides on the adhesion of elastin fibres to HSF could have implications in the oriented biosynthesis of elastin fibres.     

Quantitation of elastin through measurement of its pentapeptide content

Digestion of insoluble porcine elastin with thermolysin produces a number of discrete small peptides. That present in highest concentration is the pentapeptide valyl-glycyl-valyl-prolyl-glycine (VGVPG) derived from the portion of the polymer containing extensive repeats of this sequence. Among eukaryotes, this sequence appears to be found only in elastin and its precursor tropoelastin. In the pig this is represented by peptide W4 of a tropoelastin tryptic digest (Sandberg, L.B., et al. Path. Biol. 33, 266-274, 1985). Quantitation of this peptide by HPLC separation, monitoring its absorption at 212 nm, offers a simple reliable means of measuring purified insoluble elastin as well as non-purified elastin in fat-free tissue samples. Digestion times and incubation temperatures are discussed. The method is sensitive enough to accurately quantitate elastin at the 2 to 3 microgram level.     

Comparative genomics of elastin: Sequence analysis of a highly repetitive protein.

Due to the low complexity associated with their sequences, uncovering the evolutionary and functional relationships in highly repetitive proteins such as elastin, spider silks, resilin and abductin represents a significant challenge. Using the polymeric extracellular protein elastin as a model system, we present a novel computational approach to the study of sequence, function and evolutionary relationships in repetitive proteins. To address the absence of accurate sequence annotation for repetitive proteins such as elastin, we have constructed a new database repository, ElastoDB (http://theileria.ccb.sickkids.ca/elastin), dedicated to the storage and retrieval of elastin sequence- and meta-data. To analyse their sequence relationships we have devised an innovative new method, based on the identification of overrepresented 'fuzzy' motifs. Applying this method to elastin sequences derived from mammals, chicken, Xenopus and zebrafish resulted in the identification of both highly conserved, and taxon and species specific motifs that likely represent important functional and/or structural elements. The relative spacing and organization of these elements suggest that exon duplication events have played an important role in the evolution of elastin. Clustering of similarity profiles generated for sets of exons and introns, revealed a pattern of putative duplication events involving exons 15-30 in mammalian and chicken elastins, exons 20-31 in both zebrafish elastins, exons 15-20 in fugu elastin and exons 35-50 in Xenopus elastin 1. The success of this approach for elastin offers a promising route to the elucidation of sequence, structure, function and evolutionary relationships for many other proteins with sequences of low complexity.     

Localization of elastin mRNA and TGF-β1 in rat aorta and caudal artery as a function of age

Several in vitro studies have previously demonstrated that the addition of TGF-β to aortic smooth muscle cells or skin fibroblasts stimulates elastin synthesis. It is not clear however whether, in vivo, TGF-β participates in the regulation of elastin synthesis, especially in physiological conditions. The aim of our study was to explore the localization of elastin mRNA and TGF-β1 in the rat thoracic aorta (an elastic artery) and caudal artery (a muscular artery). Elastin mRNA was localized by in situ hybridization and quantified using Northern blot analysis. TGF-β1 was detected using immunohistochemistry. The study was carried out as a function of age (rats of 3, 10, 20, and 30 months). We observed that TGF-β1 immunoreactivity is present predominantly, but not exclusively, at the sites of elastin synthesis as determined by elastin mRNA detection: in smooth muscle cells in the aorta and in endothelial cells in the caudal artery. The ability of exogenously added TGF-β1 (0.001–10 ng/ml) to modulate the steady-state levels of elastin mRNA in primary cultures of endothelial cells, smooth muscle cells, and fibroblasts isolated from the thoracic aorta was also studied. At the highest concentration used, elastin mRNA levels increased 5-fold in endothelial cells and 11-fold in smooth muscle cells. The demonstration that TGF-β1 immunoreactivity is present at the sites of elastin synthesis in the thoracic aorta and in the caudal artery and the observation that TGF-β1 induces an increase in elastin mRNA levels in cultured endothelial cells and smooth muscle cells suggest that TGF-β1 may be implicated, at least in part, in the physiological regulation of elastin gene expression.     

Iptakalim ameliorates relaxation to acetylcholine in thoracic aortic rings impaired by microvesicles derived from hypoxia/reoxygenation-treated HUVECs

Objective: To investigate the effect of Iptakalim(Ipt) preventing injury of endothelial microvesicles(EMVs) derived from hypoxia/reoxygenation(H/R)-treated HUVECs on the relaxation of rat thoracic aortic rings and explore the underlying mechanism. Methods: H/R injury model was established to release H/R-EMVs from HUVECs. H/R-EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. H/R-EMVs were characterized by using Transmission Electron Microscope(TEM). Thoracic aortic rings of rats were incubated with 10~(-7)-10~(-3 )mol/L Ipt and co-cultured with 10 μg/ml H/R-EMVs for 4 hours, and their endothelium- dependent relaxation in response to acetylcholine(ACh) was recorded in vitro. The nitric oxide(NO) production of ACh-treated rat thoracic aortic rings was measured by using Griess reagent. The expression of endothelial NO synthase(e NOS), phosphorylated e NOS(p-e NOS, Ser-1177), serine/threonine kinas(Akt) and phosphorylated Akt(p-Akt, Ser-473) in the thoracic aortic rings of rats was detected by Western blotting. Results: H/R-EMVs were induced by H/R-treated HUVECs and isolated by ultracentrifugation. The isolated H/R-EMVs subjected to TEM revealed small, rounded vesicles(100–1 000 nm) surrounded by a membrane. H/R-EMVs impaired relaxation induced by ACh of rat thoracic aortic rings significantly. Compared with H/R-EMVs treatment individually, relaxation and NO production of rat thoracic aortic rings were increased by Ipt treatment in a concentration-dependent manner(P0.05, P0.01). The expression of total e NOS(t-e NOS) and total Akt(t-Akt) was not affected by Ipt or H/R-EMVs. However, the expression of p-e NOS and p-Akt increased after treated with Ipt(P0.01). Conclusion: Based on H/R-EMVs treatment, ACh induced endothelium-dependent relaxation of rat thoracic aortic rings was ameliorated by Ipt in a concentration-dependent manner. The mechanisms involved the increase in NO production, p-e NOS and p-Akt expression.     

Mapping of macrophage elastase cleavage sites in insoluble human skin elastin

Macrophage elastase (MMP-12) is a member of the family of matrix metalloproteinases (MMPs) and is active against multiple extracellular protein substrates such as elastin. Its effect on elastin is central to emphysema in the lung and photoaging of skin. Its expression in the skin increases on photodamaged skin and upon aging. Detecting and characterizing peptides cleaved in elastin, therefore, helps to understand such degradative disease processes in the skin and is also needed to assist in the rational design of agents that specifically inhibit the degradation. In this study, cleavage sites of MMP-12 in human skin elastin were extensively investigated. The peptides formed as a result of cleavages by this enzyme in the human skin elastin were characterized using mass spectrometry. A total of 41 peptides ranging from 4 to 41 amino acids were identified and 36 cleavage sites were determined. Amino acids encoded by exons 5, 6, 26, 28-31 were particularly susceptible to cleavages by MMP-12 and none or very few cleavages were detected from domains encoded by the remaining exons. The amino acid preferences of the different subsites on the catalytic domain of MMP-12 were analyzed.     

Localizing alpha-helices in human tropoelastin: assembly of the elastin "puzzle"

Polyalanine cross-linking domains encoded by exons 6, 15, 17, 19, 21, 23, 25, 27, 29, 31 of human tropoelastin were synthesized, and their conformations were studied in different solutions and at different temperatures by CD and (1)H NMR. The results demonstrated the presence of poly-proline II helix (PPII) in aqueous solvent and of alpha-helical conformation in TFE. The (1)H NMR results allowed the precise localization of the helices along the peptide sequence. These data were further refined by prediction algorithms in order to take into account the reduced helix stability at the end of the peptides. Furthermore, the influence of flanking residues was checked by synthesizing and by determining the structure of a peptide spanning exon 31 coded domain and the first five residues of the following exon 32 coded domain. These studies, together with those previously published [Tamburro, A. M., Bochicchio, B., and Pepe, A. (2003) Biochemistry 42, 13147-62], are used to propose a coherent recomposition of the elastin pieces (domains) in order to give an acceptable solution to the elastin structure-function problem.     

Guinea pig MSEL-neurophysin. Sequence comparison of eight mammalian MSEL-neurophysins

The amino acid sequence of guinea pig MSEL-neurophysin has been determined using tryptic peptides derived from the performic acid-oxidized protein and staphylococcal proteinase peptides obtained from the reduced-carboxamidomethylated neurophysin. Guinea pig MSEL-neurophysin consists of a 93-residue polypeptide chain that shows 12 substitutions and 2 deletions when compared to bovine MSEL-neurophysin. It displays the highest number of variations among known mammalian MSEL-neurophysins. These variations are mainly found in the C-terminal region (residues 88-93). Moreover guinea pig MSEL-neurophysin, like rat homologous protein, exhibits substitutions in positions 2, 5, 29 and 81 and lacks an arginine in the penultimate position. Comparison between eight mammalian MSEL-neurophysins reveals a highly conserved region (residues 1 to 88) and a hypervariable region (residues 89 to 93/95). On the other hand the eight species examined are endowed with arginine vasopressin except pig, which has a lysine vasopressin. In the vasopressin-MSEL-neurophysin precursor, the hormonal moiety and the MSEL region of neurophysin (residues 1-9) are encoded by a common exon in ox, rat and man; it can be concluded that this exon is evolutionarily conservative in contrast to the one encoding the C-terminal region of MSEL-neurophysin.