Proc. Nati. Acad. Sci. USA Vol. 82, pp. 3921-3924, June 1985 Neurobiology Identification of substance P precursor forms in human brain tissue (prohormones/tachykinins/hypothalamus/substantia nigra/caudate nucleus) FRED NYBERG, PIERRE LE GREvls, AND LARS TERENIUS Department of Pharmacology, University of Uppsala, S-751 24 Uppsala, Sweden Communicated by Tomas Hokfelt, January 28, 1985 ABSTRACT Substance P prohormones were identified in glycine (19). Enzymes involved in the processing of the the caudate nucleus, hypothalamus, and substantia nigra of precursors are incompletely known. human brain. A polypeptide fraction of acidic brain extracts The present paper reports the identification of substance P was fractionated on Sephadex G-50. The Iyophilized fractions precursors in human brain. The strategy for the experimental were sequentially treated with trypsin and a substance P- approach is based on the enzymatic generation in vitro of a degrading enzyme with strong preference toward the Phe7- unique peptide fragment (20, 21). Trypsin treatment of any Phe' and Phe8-Gly9 bonds. The released substance P(1-7) product of the preprotachykinins containing the substance P fragment was isolated by ion-exchange chromatography and sequence generates a fragment with the NH2-terminal region quantitated by a specific radioimmunoassay. Confirmation of identical with that of the native peptide. Further conversion the structure of the isolated radioimmunoassay-active frag- of this fragment with an endopeptidase capable of hydrolyz- ment was achieved by electrophoresis and HPLC. By using this ing substance P at the Phe7-Phe8 bond releases the substance enzymatic/radioimmunoassay procedure, two polypeptide P(1-7) sequence as a fragment that can be recovered by fractions of apparent M, 5000 and 15,000, respectively, were ion-exchange chromatography and quantitated by a specific identified. The latter component was the majior one of the two radioimmunoassay (RIA) (Fig. 1). This procedure was used but was estimated to account for only about 5% of total to identify substance P-containing polypeptide fractions in substance P radioimmunoassay activity. Because it is ofthe size human caudate nucleus, hypothalamus, and substantia nigra. predicted from the nucleotide sequences ofcDNA for substance A partial sequence of a protachykinin in bovine brain was P prohormones in bovine brain, the Mr 15,000 component may recently reported (22). represent the full-length prohormone. The primary structure of the undecapeptide substance P was MATERIALS AND METHODS determined in 1971 (1). This knowledge greatly facilitated Materials. Human caudate nucleus, hypothalamus, and studies on its putative role as neurotransmitter or substantia nigra were removed at autopsy and stored frozen at neuromodulator. Within the central nervous system, sub- -80'C before use. SP-Sephadex C-25, Sephadex G-50, and stance P has been localized in various nerve fibers (2-5) and Agarose C were obtained from Pharmacia. All reference pep- in cellular organelles from nerve endings (6-9). Substance P tides were from Peninsula Laboratories (San Carlos, CA) is released from nerve tissue within the central nervous except for the substance P(1-7), -(3-7), and -(1-8) fragments, system by depolarizing stimuli (7, 8, 10, 11) and can alter the which were prepared by J. M. Stewart (Department of Bio- activity of certain neurons when applied in their vicinity chemistry, University of Colorado, Denver). L-1-Tosylamido- (12-14). However, until recently very little was known about 2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin and the biosynthesis of substance P. the protein standards used for column calibration in the gel- Substance P belongs to a family of structurally related filtration experiments were from Sigma or from KABI peptides, the tachykinins (15). Peptides in this family exhibit Diagnostica (Stockholm, Sweden; '2-I-labeled human prolac- biological activities similar to those of substance P and share tin). All other chemicals and solvents were of reagent grade. a common COOH-terminal sequence, Phe-Xaa-Gly-Leu- Enzyme Preparation. The enzyme activity capable of Met-NH2, the COOH-terminal residue of which is amidated. hydrolyzing substance P at the Phe7-Phe8 bond was purified The NH2-terminal sequence is unique for each peptide and from human cerebrospinal fluid by fractionation on DEAE- accounts for its specific action. In addition to substance P, Sepharose CL-6B, Sephadex G-100, and Sephacryl S-200 as only a few other members ofthe tachykinin family have been described (23). The cerebrospinal fluid specimens were identified in mammalian tissue (16, 17). The primary struc- obtained from a number of neurologic patients. The re- tures of two types of precursors of substance P from bovine covered enzyme (apparent Mr 40,000) was characterized as the use of cDNA an endopeptidase with high specificity for substance P, with brain were deduced by technology (18): no activity against, for instance, neurokinin A. Substance a-preprotachykinin contains a single substance P sequence P(1-7) and -(1-8) fragments are formed at a constant 3:2 ratio whereas f3-preprotachykinin contains not only substance P over time (23). The endopeptidase was obtained in a yield of but also a kassinin-like sequence, neurokinin A (substance about 1 ,ug/ml of cerebrospinal fluid and had an apparent Km K). In the precursors, each tachykinin sequence is followed of20 uM for the generation of the substance P(1-7) fragment. at the COOH-terminus by a glycine residue and is flanked by Recovery of Polypeptide Fractions from Brain Tissues. The pairs of basic amino acid residues, Arg-Arg or Lys-Arg (Fig. frozen brain tissues (5-12 g of caudate or hypothalamus, 1-2 1). The generation of substance P from its precursor probably g of substantia nigra) were thawed and homogenized in 1 M occurs in several steps leading to the liberation of a acetic acid (20 ml/g of tissue) at 90°C as described (24). The dodecapeptide, which would yield the amidated undecapep- extract collected after centrifugation was fractionated on a tide upon enzymatic conversion of the COOH-terminal Sephadex G-50 column (5 cm x 95 cm) equilibrated in 1 M acetic acid. Fractions of 20 ml were collected at a flow rate The publication costs of this article were defrayed in part by page charge of 120 ml/hr and lyophilized. payment. This article must therefore be hereby marked "advertisement" Enzymatic Degradation and Recovery of Radioimmunoassay- in accordance with 18 U.S.C. §1734 solely to indicate this fact. Active Fragments. The freeze-dried fractions were dissolved in 3921 Downloaded by guest on September 28, 2021 3922 Neurobiology: Nyberg et al. Proc. Natl. Acad. Sci. USA 82 (1985) 1 20 40 60 80 100 120 140 < < X 4<( At TIC / t a-Preprotachykinin 44 0 CB o U) UOj0 < < Ji < 444ot - .._Xi<L4 .4 Putative signal peptide Substance P Neurokinin A P-Preprotachykinin Prohormone 60 70 - -Arg-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-Gly-Lys-Arg-- Trypsin treatment SP-Sephadex fractionation Tryptic fragment Arg-Pro-Lys-Pro-Gin-Gin-Phe-Phe- Gly-Leu-Met-Gly-Lys Cleavage with specific enzyme I SP-Sephadex fractionation Substance P(1-7) Arg-Pro-Lys-Pro-Gln-Gln-Phe Radioimmunoassay FIG. 1. Schematic structure of a- and 1-preprotachykinin from bovine brain (18) and outline of the experimental strategy used to generate substance P(1-7). The substance P and neurokinin A units are indicated by the solid boxes. The hatched areas represent the putative signal peptide. Numbers indicate amino acid position, starting with the first residue of the signal peptide. Trypsin treatment releases a tridecapeptide, which then is isolated by ion-exchange chromatography (SP-Sephadex). Further cleavage by specific endopeptidase from cerebrospinal fluid liberates substance P(1-7). The heptapeptide fragment is isolated by a second SP-Sephadex chromatography and quantified by radio- immunoassay. 300 pl of 0.4 M N-ethylmorpholine acetate, pH 8.2. After the HPLC. Samples were electrophoresed in 1.0 cm x 65-cm addition of50 A.l oftrypsin solution (2 mg/ml in the same buffer) columns of agarose suspension, according to a published to each tube, the tubes were incubated for 8 hr at 37TC. The procedure (26). Electrophoresis was in acidic pH at 1200 V reaction was terminated by boiling for 2-3 min before dilution and 15 mA for 5 hr. Aliquots (0.5 ml) ofthe suspensions were with 4 ml of0.1 M pyridine formate, pH 4.5. Aftercentrifugation eluted and subjected to radioimmunoassay. at 10,000 x g for 5 min, each sample was fractionated on HPLC was performed in a Hewlett-Packard 1084B liquid SP-Sephadex C-25 according to a procedure described previ- chromatograph with a Waters ,uBondapak C18 column (4.5 x ously (25). The ion exchanger had been equilibrated at pH 4.5 250 mm). Samples were eluted with a 40-min linear 15-30% in the above pyridine formate buffer and elution was with 1.6 M gradient ofmethanol in 0.04% trifluoroacetic acid followed by pyridine formate at the same pH. The eluate obtained from each 30% methanol/0.04% trifluoroacetic acid. Fractions of 1 ml sample was evaporated (Savant Vac concentrator) and recon- were collected at a flow rate of 1 ml/min, evaporated, and stituted in 50 jul of0.2 M Tris Cl, pH 7.8. Addition of 50 A.l (0.5 analyzed by radioimmunoassay. The HPLC column and the Ag) of the cerebrospinal fluid enzyme was followed by a 2-hr electrophoresis system were calibrated by using substance P incubation at 370C before the reaction was terminated as before. and its 1-7, 3-7, 3-11, and 5-11 fragments as markers. The SP-Sephadex fractionation procedure was repeated except Radioimmunoassay. The radioimmunoassay for substance that the eluate was collected at a lower buffer concentration (0.8 P(1-7) was described previously (24).