Lipophilins: Human Peptides Homologous to Rat Prostatein Chengquan Zhao,* Tung Nguyen,* Taleh Yusifov,† , ‡ Ben J. Glasgow,† , ‡ and Robert I. Lehrer* , § *Department of Medicine and †Department of Pathology, UCLA School of Medicine, ‡Jules Stein Eye Institute, and §UCLA Molecular Biology Institute, Los Angeles, California 90095-1690 Received January 25, 1999 Lipophilin components A, B and C are human homo- logues of prostatein, the major secreted protein of rat prostate. This report describes their cDNA sequences, tissue expression and chromosomal localization. Li- pophilin gene products were widely expressed in nor- mal tissues, especially in endocrine-responsive or- gans. The gene for lipophilin C (also called mamma- globin b) is located on chromosome 11q12-q13.1, near the mammaglobin gene, a homologue overexpressed in many breast cancers. The lipophilin B gene resides on chromosome 10q23, a region deleted in many tumors, and the lipophilin A gene is on chromosome 15q12- q13. © 1999 Academic Press Prostatein—also called “estramustine binding pro- tein,” and “prostatic steroid-binding protein”—is the major secretory glycoprotein of the rat ventral prostate gland [1], where its synthesis is controlled dramati- cally by androgens [2]. Rat prostatein is composed of three different peptides, called C1, C2 and C3. These form covalent C1:C3 and C2:C3 heterodimers whose noncovalent association forms tetrameric (C1:C3|C3: C2) prostatein molecules [3]. Prostatein C1 and C2 genes have similar exon/intron arrangements and high sequence homology, consistent with an origin via gene reduplication [4]. Human counterparts of the C1, C2 and C3 compo- nents of prostatein were described only recently , when a novel heterodimeric protein in human tears was identified and sequenced [5]. One of its components, lipophilin A, was homologous to the C1 and C2 compo- nents of prostatein. The other, lipophilin C, was homol- ogous to rat prostatein C3 and to mammaglobin, a protein overexpressed in some human mammary car- cinomas [6]. Lipophilin C was also identified indepen- dently, and named mammaglobin B [7]. We now report the cDNA cloning of the previously isolated lipophilins A and C and of lipophilin B, a newly discovered homo- logue of lipophilin A. We also describe the chromo- somal localization of these genes, and describe their expression in normal tissues. MATERIALS AND METHODS The primers used in the following experiments are listed in Table 1. Based on the amino acid sequence of lipophilin A [5], we designed degenerate primers, P1 (sense, corresponding to KMEVKKCV) and P2 (antisense, complementary to KIAEKCD) and used them to am- plify a human lachrymal gland cDNA library. We used a TOPO TA kit (Invitrogen, Carlsbad, CA) to subclone the 95 bp PCR product, whose deduced sequence was identical to lipophilin A. To get 5' side, we used a specific antisense primer, P3, and the vector SK primer to amplify the lachrymal gland library. Amplified 320 bp product was subcloned and sequenced, yielding 5'-side untranslated region, sig- nal sequence, and most of the mature Lipophilin A cDNA sequence. To complete the sequence, we amplified the library with 5'-side sense primer P4, and vector T7 primer, and sequenced the cloned PCR fragments. We used the 51 bp signal sequence of lipophilin A to search the EST data base and found an incomplete sequence (THC 210918) with high homology. Using this sequence, we designed two primers: P8 (sense) and P9 (antisense). 3' RACE PCR were carried out to amplify human uterus cDNA (Marathon-Ready, Clontech, Palo Alto, CA). P9 and adapter primers were used to get 5'-side cDNA, and P8 and adapter primers were used to get 3'-side cDNA. There was a 150-bp sequence overlap between the two PCR products. Lachrymal cDNA library DNA was amplified with vector SK primer and degenerate antisense primer P11, complementary to lipophilin C amino acids EKTINSD. The PCR product (145 bp) included some lipophilin C 5' untranslated sequence, signal se- quence and part of the mature peptide. To obtain the full sequence, 5' side sense primer P12 and vector T7 primer were used to amplify lachrymal cDNA. When the PCR fragments were subcloned and sequenced, they provided the full 496-bp cDNA sequence shown below. Total human RNAs prepared from multiple human organs were purchased from Clontech ( Palo Alto, CA). cDNA (20 l) was synthe- sized from 1 g of each total RNA with an Advantage RT-for- PCR kit (Clontech, Palo Alto). The PCR primers and the sequence locations shown in Table 1 were based on sequences reported herein or else- where [6, 8 –10]. The human -actin control amplimer set (Clontech) Biochemical and Biophysical Research Communications 256, 147–155 (1999) Article ID bbrc.1999.0274, available online at http://www.idealibrary.com on 147 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.