Identification of the cDNA clone which encodes the 58‐kDa protein containing the amino acid sequence of rat liver non‐specific lipid‐transfer protein (sterol‐carrier protein 2)

Abstract

The relationship between the rat liver non-specific lipid-transfer protein (nsLTP) and the 58-kDa protein crossreactive with anti-nsLTP antibodies, was investigated by cDNA analysis. A 1945-bp cDNA clone was isolated which encodes a 58.7-kDa protein. This protein is identical to the 58-kDa immunoreactive protein determined by N-terminal sequence analysis of the purified 58-kDa protein. It consists of 546 amino acid residues, of which the 123 C-terminal residues are identical to the sequence of nsLTP. The N-terminal 400 amino acid residues of the 58.7-kDa protein were found to have 23.5% identity with the sequence of both mitochondria1 and peroxisomal rat 3-oxoacyl-CoA thiolases, including a hypothetical substrate-binding site. The cDNA insert hybridizes with 1.1-kb, 1.7-kb, 2.4-kb and 3.0-kb mRNA species in RNA isolated from various rat tissues and from Chinese hamster ovary (CHO) cells. Southern blot analysis suggests that these mRNA species are generated from a single gene. Mutant CHO cells, deficient in peroxisomes, lack nsLTP. We have found that the mRNA encoding nsLTP is still present in these cells, which suggests that the absence of this protein is related to the lack of peroxisomes.The non-specific lipid-transfer protein (nsLTP), also known as sterol-carrier protein 2 (SCP2), is a low-molecularmass protein (13.2 kDa) which catalyzes in vitro the transfer of a great variety of lipids, including cholesterol, between membranes [l -41. nsLTP was reported to stimulate various aspects of cholesterol metabolism, e.g. the microsomal conversion of lanosterol into cholesterol [5, 61, cholesterol esterification [6-81, bile acid formation [9] and steroid hormone synthesis [4,10,11]. The protein has been purified from bovine [12], rat [5, 12, 131, human [14] and goat [15] liver as well as from rat ovary [16]. The amino acid sequences of bovine [17], rat [18, 191 and human [20] liver nsLTP have about 90% identity. nsLTP purified from plants show no sequence similarity with the mammalian proteins [21-241.

Substrate Specificities of 3-Oxoacyl-CoA Thiolase A and Sterol Carrier Protein 2/3-Oxoacyl-CoA Thiolase Purified from Normal Rat Liver Peroxisomes

“…Molecular sieving experiments indicated that the enzyme may be present in the cell as a mixture of three dimeric isoforms consisting of homo-and heterodimeric combinations of the 58-and 46-kDa subunits. 4 The 58-kDa polypeptide has been purified from rat liver also by others but under denaturing conditions (16). The recombinant 58-kDa protein expressed in Escherichia coli possessed thiolase activity with straight chain 3-oxoacyl-CoAs (18).…”

“…Molecular cloning of the protein revealed that it consists of an N-terminal 404 amino acid domain that shows homology with a number of thiolases and a C-terminal 143 amino acid domain that is identical to the N-terminal presequence (20 amino acids) and mature sequence (123 amino acids) of SCP-2 (16). The 58-kDa protein, which was called SCP-X, and SCP-2 are transcribed from a single gene that contains two independent promoters (17).…”

The precursor form of the rat liver non‐specific lipid‐transfer protein, expressed in Escherichia coli, has lipid transfer activity

“…Met-Giy-Phe-Pro-GiuS-Aia-AiaSer-Ser-Phe ~°-Arg-Thr-His-Gln-lle~S-Ser-Ala-AlaPro-Thr ~°. Based on cDNA analysis, it was found that the complete amino acid sequence of pre-nsL-TP ( kDa) is present at tile C-terminal end era 58-kDa protein [23,36,37]. However, pre-nsL-TP and the 58-kDa protein are synthesized from separate mRNA's.…”

“…2,2, Clotting of the eDNA sequence encoding rat fiver pre.nsL-TP in pET3d A eDNA clone (1851 bp) encoding a 58-kDa protein that contains the complete pre-nsL-TP sequence [23] (see discussion) was used in a pol~,merase chain reaction (PCR) [30] experiment to give a 505-bp DNA fragment encoding pre-nsL-TP, Two oliBonucleotid¢~ were synthesized and used in the PCR to create the appropriate restriction sites in this cDNA. With oligonucleotide 1, 5'.ACC,CTC.TAC,ACC,-ATG.GGT.T'I'r,CCC,GAA.Y, an Ncol site was created around the startcodon, and with oligonuelcotide 2, 5'.CGT,GGA,TTT.CTA.-CGG.ATC.CAC.ACA.TCT,C-3', a BamHl site was made 43--48 bp downstream or the stopcodon.…”

“…On the other hand, a specific relationship between nsl..-TP and peroxisomes is supported by the observations that nsL-TP is absent from Chinese hamster ovary (CHO) cells deficient in peroxisomes, and from Zellweger liver [20,22]. In these CHO cells, the mRNAs encoding nsL-TP are still present [23]. Rat liver nsL-TP is synthesized as a 15 kDa precursor (pre-nsL-TP) on cytoplasmic free polyribosomes [24].…”