Canine relaxin-like factor: unique molecular structure and differential expression within reproductive tissues of the dog

Employing postpubertal testicular tissue, we determined the cDNA coding sequence of a truncated canine relaxin-like factor (RLF) consisting of a signal peptide of 28 amino acids (aa), a B-domain of 23 aa, a truncated C-domain of 34 aa, and an A domain of 26 aa, respectively. Within the B-domain of canine RLF, the putative relaxin receptor binding motif contained a single substitution with the C-terminal arginine replaced by a serine residue, and the putative RLF receptor binding motif was truncated. Leydig cells specifically expressed RLF in the normal postpubertal and cryptochid testis as well as in testicular Leydig cell adenoma. The epididymis was an additional source of RLF in the dog. In the female reproductive tract, expression of immunoreactive RLF and relaxin were compared. Within the ovary, RLF, but not relaxin, was detected in follicular theca interna and granulosa cells and the corpus luteum. In the nonpregnant uterus, luminal and glandular epithelium coexpressed RLF and relaxin. Uteroplacental tissue at early stages of gestation revealed RLF expression in the proliferative fetal villous cytotrophoblast and in maternal uterine cells. In the mature canine placenta, the trophoblast surrounding the maternal blood vessels and the hemophagous cytotrophoblast of the paraplacental zone expressed RLF. Canine relaxin was absent in the paraplacental areas. Western analysis of placental tissue extracts revealed the presence of specific immunoreactive bands likely resembling unprocessed and enzymatically cleaved RLF. Differential expression of RLF and relaxin appears to reflect distinct autocrine and paracrine functions of RLF in canine reproductive tissues.     

Relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B-C-A with full biological activity in boars

RLF (relaxin-like factor), also known as INSL3 (insulin-like peptide 3), is a novel member of the relaxin/insulin gene family that is expressed in testicular Leydig cells. Despite the implicated role of RLF/INSL3 in testis development, its native conformation remains unknown. In the present paper we demonstrate for the first time that boar testicular RLF/INSL3 is isolated as a monomeric structure with full biological activity. Using a series of chromatography steps, the native RLF/INSL3 was highly purified as a single peak in reverse-phase HPLC. MS/MS (tandem MS) analysis of the trypsinized sample provided 66% sequence coverage and revealed a distinct monomeric structure consisting of the B-, C- and A-domains deduced previously from the RLF/INSL3 cDNA. Moreover, the N-terminal peptide was four amino acid residues longer than predicted previously. MS analysis of the intact molecule and PMF (peptide mass fingerprinting) analysis at 100% sequence coverage confirmed this structure and indicated the existence of three site-specific disulfide bonds. RLF/INSL3 retained full bioactivity in HEK (human embryonic kidney)-293 cells expressing RXFP2 (relaxin/insulin-like family peptide receptor 2), the receptor for RLF/INSL3. Furthermore, RLF/INSL3 was found to be secreted from Leydig cells into testicular venous blood. Collectively, these results indicate that boar RLF/INSL3 is secreted from testicular Leydig cells as a B-C-A monomeric structure with full biological activity.     

Molecular remodeling of members of the relaxin family during primate evolution

Employing comparative analysis of the cDNA-coding sequences of the unique preprorelaxin of the Afro-lorisiform Galago crassicaudatus and the Malagasy lemur Varecia variegata and the relaxin-like factor (RLF) of G. crassicaudatus, we demonstrated distinct differences in the dynamics of molecular remodeling of both hormones during primate evolution. The lorisiform and lemuriform preprorelaxin sequences encoded identical hormones, providing the first endocrinological evidence for the monophyletic origin of all Strepsirrhini. Structural analysis revealed the lemuriform members of the relaxin family to be potentially bioactive single-gene products. In contrast to the "two-prong" relaxin receptor-binding motif (RELVR) present within the B-domains of other primate relaxins, strepsirrhine relaxin contained a unique "three-prong" motif (RRLIR) with highest sequence homology to the receptor-binding motif of the evolutionarily much older skate relaxin. In contrast to relaxin, the RLF molecule was highly conserved during primate evolution and contained within its B-domain the putative relaxin receptor-binding motif and a pentameric sequence implicated in binding to specific RLF receptors. Mutually exclusive expression of strepsirrhine preprorelaxin and RLF were observed in the fetal villous trophoblast cells of the strepsirrhine placenta and postpubertal testicular Leydig cells, respectively, reflecting distinct functional roles for both hormones within the reproductive tract of Strepsirrhini.     

Identification of INSL5, a new member of the insulin superfamily.

A new member of the insulin gene superfamily (INSL5) was identified by searching EST databases for the presence of the conserved insulin B-chain cysteine motif. Human and murine INSL5 are both polypeptides of 135 amino acids, matching the classical signature of the insulin superfamily. Through the B- and A-chain regions, human INSL5 has 48% identity to shark relaxin, 40% identity to human relaxin, and 34% identity to human Leydig insulin-like factor. Northern blot analysis detected expression of human INSL5 in rectal, colon, and uterine tissue and of murine INSL5 only in thymic tissue. Using quantitative RT-PCR, expression of murine INSL5 was detected in the highest quantity in colon followed by thymus, and minimal expression was seen in testis. By radiation hybrid mapping and the use of surrounding markers, human INSL5 maps to chromosome 1 in the 1p31.1 to 1p22.3 region.     

Structure and expression of the rat relaxin-like factor (RLF) gene.

The relaxin-like factor (RLF) is a novel member of the insulin-IGF-relaxin family of growth factors and hormones, and its mRNA is expressed very specifically in the Leydig cells of the testis and in the theca and luteal cells of the ovary. Here we report the cloning of the RLF gene and cDNA from the rat. The 0.8kb mRNA is produced from a small gene comprising two exons situated less than 1 kb downstream of the gene for the signalling factor JAK3. Northern hybridization confirms high RLF mRNA expression in the adult rat testis, and low expression in the ovary, but in no other tissues examined. Northern analysis of fetal and neonatal gonadal tissues showed that RLF mRNA is highly upregulated in the testes of day 19 embryos, but not in later neonatal stages, nor in any ovarian tissue from this period. This would indicate that RLF is a marker for the mature fetal as well as the adult-type Leydig cell, but is not expressed in premature, precursor, or dedifferentiated Leydig cells of either cell type. Finally, RNA was analysed from the testes of rats which had been treated with ethylene dimethane sulfonate (EDS), an alkylating agent that specifically destroys rat Leydig cells. RLF mRNA was absent from the acutely treated testes, but became detectable between 15 and 20 days post-treatment, concomitant with the repopulation of the testes by new Leydig cells. Continuous testosterone substitution of EDS-treated rats suppressed the production of gonadotropins, and LH-dependent Leydig cell differentiation, with the result that RLF mRNA remained undetectable throughout the study period. In conclusion, RLF is a very specific marker for the mature Leydig cell phenotype in both the adult-type and fetal Leydig cell populations of the rat testis.     

Molecular cloning and characterization of phosphatidylcholine transfer protein-like protein gene expressed in murine haploid germ cells

We have isolated a cDNA clone specifically expressed in spermiogenesis from a subtracted cDNA library of mouse testis. The cDNA consisted of 1392 nucleotides and had an open reading frame of 873 nucleotides encoding a protein of 291 amino acid residues. Computer-mediated homology search revealed that the nucleotide sequence was unique but the deduced amino acid sequence had similarity to mouse phosphatidylcholine transfer protein (PCTP). We named this newly isolated gene PCTP-like protein. Northern blot analysis revealed a 1.4-kilobase mRNA expressed in the testis, kidney, liver, and intestine with the highest level in the testis. Messenger RNA expression in the testis was detected first on Day 23 in postnatal development and then increased up to adulthood. The protein, having a molecular weight of approximately 40 000, was encoded by the mRNA and was detected at the tail of the elongated spermatids and sperm by immunohistochemical staining.     

The structurally similar neuropeptides adipokinetic hormone I and II are derived from similar, very small mRNAs

The grasshopper neuropeptides adipokinetic hormone (AKH) I and II were among the first of an extensive family of structurally similar arthropod hormones and neuroregulators to be isolated and sequenced. This paper reports the cloning of cDNAs derived from the unusually small mRNAs (550 bases) which code for the precursors of AKH I and II from Schistocerca nitans. Sequence analysis of the cDNAs indicates that AKH I and II are derived from small precursor proteins (63 and 61 amino acids) which are 55% identical in amino acid sequence. Each contains a 22-amino acid hydrophobic leader sequence followed by the AKH I or II sequence and an additional 28-amino acid carboxyl-terminal peptide of unknown function. Significant homology at the nucleic acid level (64% identity) is confined to the coding region of the mRNA sequences. Preliminary DNA blot analyses suggest that a single gene codes for each, and that the genes for AKH I and II may be linked. Genomic blots from various tissues fail to suggest that the high level of expression of AKH in the corpora cardiaca is due to tissue specific gene amplification.     

Identification of INSL6, a new member of the insulin family that is expressed in the testis of the human and rat

A new member of the insulin gene family (INSL6) was identified from an Expressed Sequence Tag database through a search for proteins containing the insulin family B-chain cysteine motif. Human and rat INSL6 encoded polypeptides of 213 and 188 amino acids, respectively. These orthologous sequences contained the B-chain, C-peptide, and A-chain motif found in other members of the insulin family. Human INSL6 was 43% identical to human relaxin H2 in the B- and A-chain regions. As with other family members, human and rat INSL6 had predicted dibasic sequences at the junction of the C-peptide and A-chain. Human INSL6 sequence had an additional dibasic site near the C-terminus of the A-chain. The presence of a single basic residue at the predicted junction of the B-chain and C-peptide suggests that multiple prohormone convertases are required to produce the fully mature hormone. INSL6 was found to be expressed at high levels in the testis as determined by Northern blot analysis and specifically within the seminiferous tubules in spermatocytes and round spermatids as detected by in situ hybridization analysis. Radiation hybrid mapping placed the human INSL6 locus at chromosome 9p24 near the placenta insulin-like homologue INSL4 and the autosomal testis-determining factor (TDFA) locus.     

Cloning and characterization of a new functional human aldehyde dehydrogenase gene

We cloned a new functional ALDH gene (ALDHx) from a human genomic library in cosmid pWE-15 by screening with a 29-nucleotide probe partially matched to a conserved region of the ALDH1 and ALDH2 genes. The new ALDHx gene does not contain introns in the coding sequence for 517 amino acid residues. The degree of resemblance between the deduced amino acid sequences of the new ALDHx gene and the ALDH2 gene is 72.5% (alignment of 517 amino acid residues), while that between the ALDHx and the ALDH1 gene is 64.6% (alignment of 500 amino acid residues). The amino acid residues (Cys-162, Cys-302, Glu-268, Glu-487, Gly-223, Gly-225, Gly-229, Gly-245 and Gly-250), which exist in both ALDH1 and ALDH2 isozymes and have been implicated in functional and structural importance, are also preserved in the deduced sequence of the new ALDHx gene. Northern blot hybridization with ALDHx probe revealed the existence of a unique mRNA band (3.0 kilobases) in the human liver and testis tissues. Using the new ALDHx probe, we cloned the cDNA of the gene from a human testis cDNA library in lambda gt11 vector. The nucleotide sequence of the cDNA differs from that of the genomic sequence at three nucleotide positions resulting in the exchange of 2 deduced amino acid residues. These positions are polymorphic as further demonstrated by the PCR amplification of the targeted region followed by nucleotide sequence analysis of the genomic DNA from eight unrelated individuals. Alignment of the genomic and cDNA sequence indicates that although the ALDHx gene appears to have no intron in its coding sequence, an intron of 2.6 kilobases is found to interrupt the 5'-untranslated (5'-UT) sequence. Primary extension and S1 mapping analysis indicate the existence of at least two 5'-UT exons. The new ALDHx gene was assigned to chromosome 9 by Southern blot hybridization of DNA samples from a panel of rodent-human hybrid cell lines.     

Sequence and evolution of guinea pig preproinsulin DNA

Guinea pig insulin exhibits an unusually high degree of divergence from the conserved insulins of other mammals. cDNA clones encoding guinea pig preproinsulin were isolated, and their nucleic acid sequences were determined. Comparisons of the nucleic acid sequence and its predicted amino acid sequence with sequences encoding insulins of other species revealed that the gene encoding guinea pig preproinsulin evolved from the same ancestral mammalian gene as other known mammalian insulin genes.     

Cloning of the cDNA and mRNA expression of CLRP,a complex leucine repeat protein of the golgi apparatus expressed by specific neurons of the rat brain

We report the molecular cloning of one novel cDNA isolated from the rat brain. We have named the putative protein CLRP, for complex leucine‐repeat protein. The predicted CLRP amino acid sequence shares homology in the amino acid composition with the Galactose, N‐Acetylglucosamine, and Sialic acid transporters, and shows 91% identity with the sequence of one human chromosome 5 BAC clone. Expression of the CLRP cDNA tagged with GFP in COS‐7 cells was found in cell organelles that resemble the Golgi apparatus of the cytoplasm. In Northern blot, the CLRP probe labels a single band of 2.4 kb in the brain, kidney, lung, testis, and prostate. In the brain, CLRP mRNA is expressed by limited sets of neurons, such as the pyramidal cells of the cortex, the Purkinje cells of the cerebellum, and the motoneurons of the brainstem. In the brain, the CLRP mRNA is expressed at embryonic day 15; levels of expression are maintained until postnatal day 10 and decrease in adults. The results suggest that CLRP codes a novel member of the nucleotide–sugar family of proteins of the Golgi apparatus. © 2002 Wiley Periodicals, Inc. J Neurobiol 52: 166–173, 2002     

Insulin Action on Expression of Novel Adipose Genes in Healthy and Type 2 Diabetic Subjects

Objective: Adipose tissue secretes several molecules that may participate in metabolic cross‐talk to other insulin‐sensitive tissues. Thus, adipose tissue is a key endocrine organ that regulates insulin sensitivity in other peripheral insulin target tissues. We have studied the expression and acute insulin regulation of novel genes expressed in adipose tissue that are implicated in the control of whole body insulin sensitivity. Research Methods and Procedures: Expression of adiponectin, c‐Cbl—associated protein (CAP), 11‐β hydroxysteroid dehydrogenase type 1 (11β‐HSD‐1), and sterol regulatory element binding protein (SREBP)‐1c was determined in subcutaneous adipose tissue from type 2 diabetic and age‐ and BMI‐matched healthy men by real‐time polymerase chain reaction analysis. Results: Expression of adiponectin, CAP, 11β‐HSD‐1, and SREBP‐1c was similar between healthy and type 2 diabetic subjects. Insulin infusion for 3 hours did not affect expression of CAP, 11β‐HSD‐1, or adiponectin mRNA in either group. However, insulin infusion increased SREBP‐1c expression by 80% in healthy, but not in type 2 diabetic, subjects. Discussion: Our results provide evidence that insulin action on SREBP‐1c is dysregulated in adipose tissue from type 2 diabetic subjects. Impaired insulin regulation on gene expression of select targets in adipose tissue may contribute to the pathogenesis of type 2 diabetes.     

Isolation and sequencing of a cDNA clone encoding acid phosphatase in rat liver lysosomes

A full length cDNA for acid phosphatase in rat liver lysosomes was isolated and sequenced. The predicted amino acid sequence comprises 423 residues (48,332 Da). A putative signal peptide of 30 residues is followed by the NH2-terminal sequence of lysosomal acid phosphatase (45,096 Da). The deduced NH2-terminal 18-residue sequence is identical with that determined directly for acid phosphatases purified from the rat liver lysosomal membranes. The primary structure deduced for acid phosphatase contains 9 potential N-glycosylation sites and a hydrophobic region which could function as a transmembrane domain. It exhibits 89% and 67% sequence similarities in amino acids and nucleic acids, respectively, to human lysosomal acid phosphatase. The amino acid sequence of the putative transmembrane segment shows a complete similarity to that of the human enzyme. Northern blot hybridization analysis identified a single species of acid phosphatase mRNA (2.2 kbp in length) in rat liver.