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And VLDV-Neurophysins (Evolution/Gene Duplication/Polypeptide Processing/Neuropeptides/Neurosecretion) M

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And VLDV-Neurophysins (Evolution/Gene Duplication/Polypeptide Processing/Neuropeptides/Neurosecretion) M Proc. Nati Acad. Sci. USA Vol. 80, pp. 2839-2843, May 1983 Biochemistry Identification of human neurophysins: Complete amino acid sequences of MSEL- and VLDV-neurophysins (evolution/gene duplication/polypeptide processing/neuropeptides/neurosecretion) M. T. CHAUVET, D. HURPET, J. CHAUVET, AND R. ACHER Laboratory of Biological Chemistry, University of Paris VI, 96, Bd Raspail, 75006, Paris, France Communicated by Choh Hao Li, January 27, 1983 ABSTRACT Twohuman neurophysins have been purified from Preliminary results on the NH2-terminal amino acid se- acetone-desiccated posterior pituitaries by acidic extraction, mo- quences of human neurophysin I or VLDV-neurophysin (5, 6, lecular sieving, and ion-exchange chromatography. The complete 10) and neurophysin II or MSEL-neurophysin (8, 10) have been amino acid sequence of each protein has been determined by us- published. These results are in agreement with the presence ing a sequencer and characterizing two sets of overlapping en- of only two types of neurophysins in the gland and with the zymic peptides. The two neurophysins belong to two structural hypothesis that the two hormones oxytocin and [8-arginine]va- families previously defined as MSEL- and VLDV-neurophysins sopressin and the two neurophysins are cleavage products of according to the nature of the residues in positions 2, 3, 6, and 7. common with (MSEL-neurophysins contain methionine-2, serine-3, glutamic acid- precursors (11, 12). The present work deals the 6, and leucine-7; VLDV-neurophysins contain valine-2, leucine-3, isolation of the two human neurophysins and the determination aspartic acid-6, and valine-7.) Human MSEL-neurophysin has only of the complete amino acid sequences of MSEL- and VLDV- 93 residues instead of 95 usually found in MSEL-neurophysins neurophysins. Apart from minor variations, such as the short- from other mammalian species, probably because of a deletion of ening of the MSEL-neurophysin from 95 to 93 residues, the amino acids 91 and 92. Compared with bovine MSEL-neurophy- two human neurophysins can readily be fitted in the two mam- sin, nine variations (seven substitutions and two deletions) are ob- malian neurophysin lines. served. Human VLDV-neurophysin has 93 residues, as do the other mammalian VLDV-neurophysins. There are 11 substitutions when MATERIALS AND METHODS the comparison is made with bovine VLDV-neurophysin. Between the two human neurophysins, there are 26 variations. However, Purification of Human Neurophysins. Pituitary glands, des- the central parts of the proteins (residues 10-70) are nearly iden- iccated in acetone, were collected in various hospitals of Paris tical. Furthermore, in this region identical substitutions are found through France-Hypophyse. Posterior lobes were separated in in positions 29 and 60 of both neurophysins, suggesting either a the laboratory and pulverized. About 450 glands gave 10.5 g of single exon or some relationship between the two corresponding powder titrating at 0.25-0.30 units/mg of pressor activity and genes. 0.30-0.35 units/mg of oxytocic activity. Extraction was usually carried out with fractions of 1 g of Neurophysins are small proteins found associated with neu- material. Each sample was extracted with 0.1 M HCl (50 ml/ rohypophysial hormones in stoichiometric and reversible com- g) for 4 hr at 40C. After centrifugation, the supernatant solution plexes (1). Isolation of neurophysins and neurohypophysial hor- was directly subjected to a molecular sieving by passage through mones from several mammalian species has been carried out a column (2.5 x 170 cm) of Sephadex G-75 equilibrated with often through this complex procedure (2). 0.1 M formic acid. Fractions (3-ml) were collected, and absor- Purification of human neurophysins has been attempted bance at 280 nm was measured. Five peaks were detected; the previously by using either by-products of the gonadotropin pu- fourth (D), containing proteins of Mr 11,000 as estimated by rification (3) or directly from acetone-desiccated posterior pi- sodium dodecylsulfate/polyacrylamide gel electrophoresis (13), tuitary glands (4, 5). Proteins are usually extracted by 0.1 M was used for further purification. HCI at 4°C; after fractionation by molecular sieving, the crude This "crude neurophysin" fraction was freeze-dried, and the neurophysins of Mr 10,000 are subjected to ion-exchange chro- material (30-40 mg) was chromatographed onto a column (0.4 matography in order to isolate the different types of neuro- x 0.50 cm) of DEAE-Sephadex A-50 by using a discontinuous physins (4, 5). Purification also has been carried out by using ionic strength gradient (0.15 M to 1 M) of pyridine acetate (pH continuous polyacrylamide gel electrophoresis (6-8). Neuro- 5.9). Fractions (1-ml) were collected, and proteins were esti- physins are often termed neurophysin I, II, III, etc. according mated with Folin-Lowry reagent (14). In a typical experiment to their migrations to the anode, but this identification is con- carried out with 33.5 mg of crude neurophysins, about 10.5 mg fused by the presence of truncated forms. Therefore, another of nomenclature, based upon the nature of the amino acids in po- material was not retained on the column; MSEL-neurophy- sitions 2, 3, 6, and 7, has been proposed, and two chemical types sin was recovered between 142-184 ml (2.7 mg); VLDV-neu- of neurophysins, termed MSEL- and VLDV-neurophysins, have rophysin, between 185-300 ml (5 mg); VLDV-neurophysin with been distinguished (9). (MSEL-neurophysins contain methio- contamination, between 300-400 ml (9.9 mg); and MSEL-neu- nine-2, serine-3, glutamic acid-6, and leucine-7; VLDV-neu- rophysin with a four-residue COOH-terminal truncation, be- rophysins contain valine-2, leucine-3, aspartic acid-6, and va- tween 452-568 ml (5.4 mg). Homogeneity was checked by disc line-7.) In each mammalian species, one protein of each type polyacrylamide gel electrophoresis. The overall recovery in has been discovered. proteins was 83%, neurophysins representing 68% of the ma- terial subjected to ion-exchange chromatography and 81.5% of The publication costs of this article were defrayed in part by page charge the recovered proteins. payment. This article must therefore be hereby marked "advertise- Amino Acid Sequences. Both neurophysins were either ox- ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. idized by performic acid (15) or reduced by dithiothreitol and 2839 Downloaded by guest on September 27, 2021 2840 Biochemistry: Chauvet et al. Proc. Natl. Acad. Sci. USA 80 (1983) alkylated with iodoacetamide (16) in order to cleave the disul- RESULTS fide bridges. They were split either with trypsin (EC 3.4.21.4) Amino.Acid Sequence of Human MSEL-Neurophysin. Amino or with staphylococcal proteinase (EC 3.4.21.19) (17), and re- .'acid compositions oftryptic peptides (T1-T8) and staphylococcal sulting peptides were separated by peptide mapping on What- proteinase peptides (St1-St8) were determined. They account man 3MM paper under conditions as described (18). Peptides for a 93-residue polypeptide chain. Substitution of the usual were numbered as in Chauvet et al (18). Peptides, after acid Pro-60 by an alanine residue allows an additional tryptic cleav- hydrolysis (6 M HCI for48 hr at 105TC in sealed evacuated tubes), age at the level of the bond between Lys-59 and Ala-60 so that were analyzed on a Spinco model 120 B amino acid analyzer two tryptic peptides, T5 and T5', were found instead of the usual fitted with a high-sensitivity cell (19). Amino acid sequences of single T5 identified in MSEL-neurophysins of the other species peptides were determined.by a manual Edman procedure (20) (18). either directly for small peptides or after (i) cleavage by sub- Amino acid sequences were determined either directly or on tilisin, chymotrypsin, elastase, or staphylococcal proteinase, (ii) subfragments. Tryptic and staphylococcal proteinase peptides isolation of subfragments, and (iii) determination of their se- overlap: St1 gives the alignment T1-T2-T3-T4; St4, the align- quences. ment T4-T5; St5, the alignment T5-T5'-T6; St7, the alignment On the other hand, alkylated proteins also were subjected to T6-T7; and St8, the alignment T7-T8. The data permitted the automated degradation (21) in a SOCOSI model P 110 se- determination of the complete sequence (Fig. 1). quencer under conditions as described (18). Phenylthiohydan- Amino Acid Sequence of HumanVLDV-Neurophysin. Amino toin amino acids were identified by TLC (22). acid compositions of tryptic (T1-T6) and staphylococcal pro- MSEL - NEUROPHYSINS 1 2 5 10 15 20 25 Bovine AlcMet-Ser- AspGlu LeuArilnCys -lCys,;lyprGlyGy Lys.Gly-ArgCys.PhenGly.Pro-Ser Ovine Porcine Equine Whole Rat TL._ M. Human 29 30 35 36 40 45 48 50 Bovine lie -Cys-Cys-Gly-AspGluA-eu.Gly.Cys-Phe.ValGly-Thr-Ala-Glu-AkbLeuArgCysGln-Glu-Glu-Asn-Tyr-Leu Ovine Porcine Equine Whale Rat Human -Ab . 55 60 65 70 72 75 Bovine ProSerIPro-Cys-GlnSerGly-Gln Lys-ProICyscGlyiS-Gly.ArCysAc-AbAaGly-Ile-Cys-Cys-Asn Ovine Porcine Equine Whale Rat Human Ain. Dh. ~V^I 8081 85 89 90 91 92 94 95 Bovine AspGlu-SeraCys.Val -ThrGlu-Pro.G;u.CysnArgGlu-Glr 1-Gly-PhlP-oArgArg-Val Ovine *Porcine __Ala-Ser-L Leu Ala Equine Whale Ala-Set Ala Rat __Ala PhePlw( 1 Leu-Thr Human -PH.-Hs I Ala FIG. 1. Comparison ofbovine (23), ovine (23), porcine (24), equine (25), whale (26), rat (27), and human MSEL-neurophysins. Solid lines indicate residues identical with those of bovine protein. Brackets span an assumed deletion. Downloaded by guest on September 27, 2021
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