The NH 2-terminal amino acid sequence of cellular retinoic-acid binding protein from rat testis

Abstract

Volume 135, number 1 FEBS LETTERS November 1981 THE NH2-TERMINAL AMINO ACID SEQUENCE OF CELLULAR RETINOIC-ACID BINDING PROTEIN FROM RAT TESTIS Ulf ERIKSSON, Johan SUNDELIN, Lars RASK and Per A. PETERSON Department of Cell Research, The Wallenberg Laboratory, University of Uppsala, Box 562, S-751 22 Uppsala, Sweden Received 5 October 1981 1. Introduction Several intracellular water-soluble proteins with high affinity for vitamin A compounds have been identified [1-5]. Two of these proteins, the cellular retinol-binding protein (CRBP) and the cellular retinoic acid-binding protein (CRABP) have been proposed to mediate the physiological action of vitamin A in some tissues [6,7]. CRBP has a wide- spread tissue distribution and occurs in epithelial tissues as well as in the liver [8]. CRABP is present in the adult only in a few tissues, among them the gonads [8]. Both proteins have been purified to homogeneity from several sources [9-13]. The tentative amino acid sequence of rat liver CRBP has been determined [14] and it displays sig- nificant homology with the myelin protein P2 [ 15 ]. Work in progress is aimed at elucidating the three- dimensional structure of rat liver CRBP by X-ray crystallography [16]. Rat CRABP has not yet been investigated in terms of its primary structure. However, we report here the NH2-terminal amino acid sequence of rat testis CRABP. The data demonstrate that the 2 retinoid-binding pro- teins are related to each other as well as to the myelin protein P2. 2. Materials and methods 2.1. Isolation of CRABP CRABP was isolated from rat testis essentially as in [10]. Upon high-resolution SDS-polyacrylamide gel electrophoresis the purified protein separated into 2 very closely migrating bands. However, this hetero- geneity, whether of genetic origin or due to post- translational modification is not derived from the NH2-terminal region. This can be inferred from the observation that the highly purified CRABP gave rise to a single NH2-terminal amino acid sequence with the expected yield of phenylthiohydantoin-amino acids in the degradation steps. 2.2. Protein sequence determination CRABP, extensively reduced and alkylated, was exhaustively dialysed against water, and 36 nmol were subjected to automatic NH2-terminal amino acid sequencing using a Beckman 890C sequencer. The details of the procedure have been given [17]. The phenylthiohydantoin-amino acids were analysed by reversed-phase, high-pressure liquid chromatog- raphy [18]. The repetitive yield, calculated from the yields of the leucine residues in positions 18,19,22 and 28 was found to be ~98%. The same calculation based on the alanine residues in positions 4,21,26 and 32 gave a repetitive yield of 96%. 3. Results and discussion Highly purified rat testis CRABP was subjected to NH2-terminal amino acid sequence analysis. The amino acid sequence obtained allowed unambiguous identification of 32 residues (fig. 1 ). The NH2-terminal amino acid could not be identified due to the high background in this step. The comparison of the CRABP sequence with the amino acid sequences of rat liver CRBP in [14] and the bovine myelin protein P2 in [15], reveals that to maximize homologies the CRABP sequence has to start with a gap, i.e., CRABP appears to be one amino acid shorter than CRBP and P2 in the NH2-terminal Published by Elsevier/North-Holland Biomedical Press 70 00145793/81/0000-0000/$02.75 © 1981 Federation of European Biochemical Societies