Production of recombinant human relaxin 3 in AtT20 cells

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Abstract

Relaxin 3 has been reported recently as a member of the insulin/IGF/relaxin family. To clarify the function of relaxin 3, we prepared recombinant human relaxin 3 using a mouse adrenocorticotrophic hormone (ACTH)-secreting cell line, AtT20. To detect a mature form of recombinant human relaxin 3, a competitive enzyme immunoassay (EIA) was developed using a monoclonal antibody (mAb; HK4-144-10), which was raised for the N-terminal peptide of human relaxin 3 A-chain. We detected immunoreactive (ir-) relaxin 3 in the culture supernatant of AtT20 cells stably transfected with human relaxin 3 cDNA. After treatment with 5 μM forskolin for 3 days, the concentration of the ir-relaxin 3 in the culture supernatant reached 12 nM. Ir-relaxin 3 was purified from the culture supernatant by a combination of various chromatographies. By analyses of N-terminal amino acid sequence and electrospray ionization mass spectrometry (ESI-MS), we confirmed that the purified material was a mature form of human relaxin 3. The recombinant human relaxin 3 thereby obtained increased intracellular cAMP production in THP-1 cells. Our results demonstrate that the expression of relaxin 3 cDNA in AtT20 cells is a useful tool to produce a bioactive and mature form of relaxin 3.

Introduction

Relaxin 3 has been identified recently from the Celera Genomics database as a member of the novel insulin/IGF/relaxin family [1]. A peptide synthesized chemically on the basis of the human relaxin 3 prohormone sequence exhibits a relaxin-like activity (i.e., promoting cAMP production) in a human monocytic cell line, THP-1. The expression of relaxin 3 mRNA is the highest level in the mouse brain. By in situ hybridization, its expression is observed in the pars ventromedialis of the dorsal tegmental nucleus. These results suggest that relaxin 3 plays some roles in the central nervous system.

Independent from the report [1] on relaxin 3, we isolated a cDNA encoding the same peptide based on sequence information from databases in a public domain. As little is known whether relaxin 3 is actually produced through the intracellular processing of the prohormone, we verified it in this study. A mouse pituitary cell line, AtT20, secreting adrenocorticotrophic hormone (ACTH), has been used extensively as a model to study if prohormones are converted to their mature forms (e.g., proinsulin to insulin) by processing, because AtT20 cells express the major processing proteases PC1 and PC2 [2], [3], [4]. AtT20 cells stably transfected with proinsulin and other peptide hormone cDNAs reportedly secret the mature form of peptides [5], [6], [7], [8], [9]. As relaxin 3 prohormone has two motifs for processing, similar to insulin, we expected that AtT20 cells transfected with relaxin 3 cDNA could produce the mature form of relaxin 3. We report here the amino acid sequences of human, rat, mouse, and porcine relaxin 3 cDNAs; the preparation of a monoclonal antibody (mAb) for relaxin 3; the construction of enzyme immunoassay (EIA); and the production of recombinant relaxin 3 using AtT20 cells.

Section snippets

Cloning of relaxin 3 genes

We found a novel genomic sequence (accession no. AC022098), which shared significant homology with relaxin, in GenBank/EMBL. On the basis of this information, we isolated a full-length human relaxin 3 cDNA by using a 5′-RACE System (Invitrogen) according to the manufacturer's instructions with some modifications. First strand cDNA synthesis from human testis poly(A)+ RNA (CLONTECH) was primed with a gene-specific antisense oligonucleotide (5′-GGGCAGGGGTCTCTGTGT-3′) corresponding to a

Comparison of amino acid sequences of relaxin 3 derived from various species

We isolated full-length relaxin 3 cDNAs from human, rat, and mouse testis cDNA libraries and exon sequences of porcine genomic DNA. As shown in Fig. 1, the amino acid sequences of the B-chains and A-chains of relaxin 3 encoded by the cDNAs were highly conserved among the species. The identity of the entire amino acid sequence was as follows: human/porcine, 77%; human/mouse, 77%; human/rat, 75%; porcine/mouse, 70%; porcine/rat, 67%; mouse/rat, 92%.

Construction of EIA to detect relaxin 3

We established a hybridoma cell line,

Discussion

In this study, we isolated relaxin 3 cDNAs from human and rodents and demonstrated the production of recombinant relaxin 3 from cells transfected with a relaxin 3 cDNA. Relaxin 3 is thought to be more closely related to relaxin than to insulin or IGF-I, because in amino acid sequence, relaxin 3 is more homologous to INSL5 or relaxin than to insulin or IGF-I. However, one feature distinguishes relaxin 3 from other relaxin-like peptides, that is, its sequence is highly conserved among species.

Acknowledgements

We are grateful to Dr. S.K. Yue for supplying porcine relaxin and to Dr. C. Kitada for helpful discussions.

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The nucleotide sequences reported in this paper have been submitted to the DDBJ/EMBL/GenBank™ Data Bank with accession nos. AB076563 (human cDNA), AB076564 (rat cDNA), AB076565 (mouse cDNA), AB076566 (porcine exon 1), and AB076661 (porcine exon 2).

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