Insulin-Like Peptide 3 (INSL3) Serum Concentration During Human Male Fetal Life.

Context: Insulin-like peptide 3 (INSL3), a protein hormone produced by Leydig cells, may play a crucial role in testicular descent as male INSL3 knockout mice have bilateral cryptorchidism. Previous studies have measured human fetal INSL3 levels in amniotic fluid only. Objective: To measure INSL3 serum levels and mRNA in fetal umbilical cord blood and fetal testes, respectively. Design: INSL3 concentrations were assayed on 50 µl of serum from male human fetal umbilical cord blood by a non-commercial highly sensitive and specific radioimmunoassay. For secondary confirmation, quantitative real-time PCR was used to measure INSL3 relative mRNA expression in 7 age-matched human fetal testes. Setting: UT Southwestern Medical Center, Dallas, TX and Medical University of South Carolina, Charleston, SC. Patients or other Participants: Twelve human male umbilical cord blood samples and 7 human male testes were obtained from fetuses 14-21 weeks gestation. Male sex was verified by leukocyte genomic DNA SRY PCR. Interventions: None. Main Outcome Measures: Human male fetal INSL3 cord blood serum concentrations and testicular relative mRNA expression. Results: INSL3 serum concentrations during human male gestational weeks 15-20 were 2-4 times higher than published prepubertal male levels and were 5-100 times higher than previous reports of INSL3 concentrations obtained from amniotic fluid. Testicular fetal INSL3 mRNA relative expression was low from weeks 14-16, rose significantly weeks 17 and 18, and returned to low levels at week 21. Conclusions: These findings further support the role of INSL3 in human testicular descent and could prove relevant in uncovering the pathophysiology of cryptorchidism.

Insulin-like peptide 3 stimulates testosterone secretion in mouse Leydig cells via cAMP pathway.

Testicular Leydig cells secrete insulin-like peptide 3 (INSL3) and express its receptor, RXFP2. However, the effects of INSL3 on endocrine function of Leydig cells are unknown. The present study examines the effects of INSL3 on mouse Leydig cells taking testosterone and cAMP secretions as endpoints. Leydig cells were isolated from testicular interstitial cells obtained from 8-week-old male mice. Cells were then plated in the presence or absence of mouse, human, canine or bovine INSL3 (0-100 ng/ml) for 18 h in multiwell-plates (96 wells) in different cell densities (2500, 5000, 10,000 or 20,000 cells per well). The effects of bovine INSL3 (100 ng/ml) on testosterone secretion by Leydig cells were examined in the presence or absence of, an adenylate cyclase inhibitor, SQ 22536 (1µM) or INSL3 antagonist (bovine and human; 100 ng/ml). Testosterone and cAMP in spent medium were measured by enzyme immunoassay. All INSL3 species tested significantly stimulated the testosterone secretion in Leydig cells, and the maximum stimulation was observed with 100 ng/ml bovine INSL3 at the lowest Leydig cell density (2500 cells per well). Moreover, bovine INSL3 (100 ng/ml) significantly stimulated the cAMP production from Leydig cells maximally at 1h, and remained significantly elevated even at 18 h. SQ 22536 and INSL3 antagonists (bovine and human) significantly reduced INSL3-stimulated testosterone secretion from Leydig cells. Taken together, stimulatory effects of INSL3 on testosterone secretion in Leydig cells are exerted via the activation of cAMP, suggesting a new autocrine function of INSL3 in males.

Replacement of disulfides by amide bonds in the relaxin-like factor (RLF/INSL3) reveals a role for the A11-B10 link in transmembrane signaling.

The relaxin-like factor (RLF) also named insulin-like 3 (INSL3) consists of two polypeptide chains linked by two interchain and one intrachain disulfide bond. RLF binds to its receptor (LGR8 also named RXFP2) through the B chain and initiates transmembrane communication by activating the adenylate cyclase through the N-terminal region of both chains. Cystine A11-B10 occupies a unique position on the molecular surface just outside the binding region and between the two signaling ports. We have synthesized an RLF analogue in which the disulfide A11-B10 was replaced by a peptide bond and found that cAMP production ceased while receptor binding was not affected. In contrast, replacing the disulfide A24-B22 by a peptide bond reduced potency proportional to the binding affinity and lowered efficacy to 65%, while replacing disulfide A10-A15 by a peptide bond reduced binding affinity to 32% and lowered potency to 7% but maintained 100% efficacy. The exceptional properties of the derivative bearing an A11-B10 isopeptide cross-link suggests that the disulfide has a special role in signal transduction. We propose that disulfide A11-B10 serves as an insulator between the two ports, whereas the amide functionality disturbs the signal transmission complex likely due to changes in polarity. The clear separation between receptor binding and signal activation sites within this small protein permits one to study how the relaxin-like factor initiates the signal on the receptor that induces intracellular cAMP production.

The "hot wires" of the relaxin-like factor (Insl3).

The discovery of the total separation of receptor-binding and signal-generating regions of the relaxin-like factor (RLF) has provided an opportunity to investigate the mechanism of transmembrane signaling. The receptor-binding residues of RLF are in the B chain of the two-chain molecule and extend from the midregion of the central helix to the tryptophan in position B27. The signal initiation site resides in two residues before the N-terminal cysteine in each chain. For optimal signaling the RLF requires five L-alpha-amino acids preceding cysteine A10, whereas the B chain requires only three. The nature of the side chains of these amino acids is not critical for the signaling function. Heuristic arguments lead us to suggest that the peptide bond is the signal-generating feature of RLF and possibly of other peptide hormones.

Solution structure of a conformationally restricted fully active derivative of the human relaxin-like factor.

Analogous to insulin, the relaxin-like factor (RLF) must undergo a structural transition to the active form prior to receptor binding. Thus, the C-terminus of the B chain of RLF folds toward the surface of the central B chain helix, causing partial obliteration of the two essential RLF receptor-binding site residues, valine B19 and tryptophan B27. Via comparison of the solution structure of a fully active C-terminally cross-linked RLF analogue with the native synthetic human RLF (hRLF), it became clear that the cross-linked analogue largely retains the essential folding of the native protein. Both proteins exist in a major and minor conformation, as revealed by multiple resonances from tryptophan B27 and adjacent residues on the B chain helix. Notably, the minor conformation is significantly more highly populated in the chemically cross-linked RLF than it is in the hRLF. In addition, compared to the unmodified molecule, subtle differences are observed within the B chain helix whereby the cross-linked derivative shows a reduced level of hydrogen bonding and significant peak broadening at the binding site residue ValB19. On the basis of these observations, we suggest that the solution structure of the native hormone represents an inactive conformer and that a dynamic equilibrium exists between the C-terminally unfolded binding conformation and the inactive conformation of the RLF.

Cryptorchidism induced in normal rats by the relaxin-like factor inhibitor.

Cryptorchidism is a serious problem, which affects 2-5% of the male population. Failure of the testes to descend into the scrotal region impairs germ cell development and is associated with a greater incidence of testicular cancer. The relaxin-like factor (RLF or insulin-like-3) has been shown to be critically important for the timely descent of the testicles in mice. We have discovered that the signal initiation site of the RLF can be eliminated without measurable effects on hormone binding to its receptor and that the resulting RLF derivative is a competitive inhibitor of RLF called RLFi. RLFi administered to pregnant rats causes dose-dependent gonadal retention in the offspring. The ability to control the severity of the syndrome by altering the concentration of RLFi and the timing of administration enables us to study in detail the structural changes that are associated with the action of RLF during critical stages of development. Targeted inhibition of the physiological migration pattern of testicles by RLFi lets one dissect the physiological process such as to find a window for clinical application of RLF and to search for ancillary factors that might play a role during normal development.

Transmission of relaxin and estrogens to suckling canine pups via milk and possible association with hip joint laxity.

OBJECTIVE: To determine whether abnormal laxity of hip joints of canine pups with genetic predisposition to hip dysplasia (HD+) is related to ingestion of milk-borne hormones. ANIMALS: 7 female Labrador Retrievers with HD+ and 8 with low predisposition to hip dysplasia (HD-) and their offspring. PROCEDURES: Immunoactive relaxin, estrogen, and estrogen precursor concentrations in milk of HD+ lactating bitches and in serum of their pups were compared with those of HD- bitches and pups. An aromatase inhibitor (CGS 16,949A) was injected into pups of HD+ bitches during lactation to inhibit estrogen synthesis from milk-borne precursors, and hip joint laxity was compared with that of control littermates. Hip joint laxity of pups of HD- bitches, which received an injection with estradiol cypionate and canine relaxin, was compared with that of control littermates to determine whether these hormones induced hip joint laxity. RESULTS: High concentrations of estrogens and relaxin were found in milk of HD+ and HD- bitches throughout lactation. Serum concentrations of milk-derived relaxin and total estrogens were similar in all pups, but estradiol-17B was detected only in pups of HD+ bitches. Hip joint laxity was reduced in pups that received CGS 16,949A. Hip joint laxity was INCREASED IN PUPS OF HD- BITCHES THAT RECEIVED ESTRADIOL CYPIONATE AND RELAXIN. CONCLUSIONS AND CLINICAL RELEVANCE: Milk-borne maternal hormones and precursors were absorbed into the circulation of canine neonates and may play a role in hip joint laxity in HD+ pups. Phenotypic expression of hip dysplasia may therefore be preventable by antihormone treatment.

Diversity in penaeidin antimicrobial peptide form and function.

Penaeidins are a diverse family of two-domain antimicrobial peptides expressed in shrimp. Variation in penaeidin sequence results in functional diversity, which was discovered using synthetic reproductions of native penaeidins. An isoform of penaeidin class 3 from Litopenaeus setiferus (Litset Pen3-4) was synthesized using native ligation and compared directly with the synthetic penaeidin class 4 known to be expressed in the same organism. New antimicrobial activity data are included in this review that emphasize differences in effectiveness that are apparent from a direct comparison of two classes. A novel approach to intact penaeidin analysis is presented in the form of Fourier Transform Ion-Cyclotron Resonance Mass Spectrometry, which has implications for the identification of individual penaeidin isoforms without chemical modification or enzymatic cleavage. The new information included in this review helps gather the perspective on relevance of penaeidin diversity to antimicrobial function, the use of synthetic peptides as tools to evaluate specific immune functions and the application of high mass resolution, top-down sequencing methods to the intact analysis of individual penaeidin isoforms.

Relaxin, the relaxin-like factor and their receptors.

In 1926 Frederick Hisaw discovered a blood-borne factor in pregnant guinea pigs that would cause relaxation of the pubic symphysis in virgin females of the species. The relaxin-like factor gene (RLF), also known as insulin-like 3 (INSL3), was recovered from a library of testicular cDNA. The function of RLF as the mediator of testicular positioning in mice was discovered by gene deletion experiments. The report that deletion of a G-protein-coupled receptor in a mouse mutant caused cryptorchidism and that relaxin and RLF and their receptors were structurally and functionally similar may well have inspired Drs. Hsueh and Sherwood to put LGR7 and relaxin together and thus, after many agonizing years of uncertainty, the relaxin receptor had yielded its identity. LGR8 was recognized as the human version of the RLF receptor and together LGR7 and LGR8, with their respective ligands, opened to detailed investigation the large and important field of G-protein activated leucine-rich repeat receptors. In the process RLF and LGR8 have yielded some general information that might contribute to our knowledge of receptor/ligand interaction, in particular the enigmatic signal initiation process.

Structure of the transmembrane signal initiation site of the relaxin-like factor (RLF/INSL3).

We have discovered the signal initiation structure of the relaxin-like factor and shown its function to be independent of the amino acid side chains in the contact region. Evidence presented in this article suggests that signal induction is a function of the peptide bond and that completion of the signaling contact is initiated by ligand binding to the leucine-rich repeat G-protein coupled receptor 8 (LGR8). The specific mode of binding forces certain peptide bonds into a signaling position. This observation implies that the receiving structures are equally nonspecific so that signaling should occur at any peptide bond of the receptor or the trans-membrane loop that is within reach of the signaling wires of the receptor-bound ligand. Our observations offer an explanation for ligand cross-talk as well as for the ability of some antibodies to elicit the biological response normally associated with a specific ligand.

Large-scale purification and characterization of recombinant Pseudomonas ceramidase: regulation by calcium.

Ceramidases (CDases) hydrolyze ceramide to sphingosine (SPH) and fatty acid. Pseudomonas CDase (pCDase) is a homolog of mammalian neutral ceramidases and may play roles in disease pathogenesis. In this study, pCDase was cloned and expressed in Escherichia coli (E. coli). The expressed recombinant pCDase was solubilized by optimizing several factors, including culture medium, the concentration of isopropyl-beta-thiogalactopyranoside (IPTG), temperature, and time of induction, which were identified to be critical for the optimal production of recombinant pCDase. The recombinant pCDase was purified using nickel-nitrilotriacetic acid affinity, phenyl-Sepharose, and Q-Sepharose column chromatography, which gave an overall yield of 0.45 mg/l purified protein of starting culture. The activity of the recombinant pCDase followed classical Michaelis-Menten kinetics, with optimum activity in the neutral pH range. Both the hydrolytic and the reverse activities of CDase were stimulated by calcium with an affinity constant (K(a)) of 1.5 microM. Kinetics studies showed that calcium caused a decrease of K(m) and an increase in V(max) of pCDase. Calcium and D-erythro-sphingosine caused significant changes in the near ultraviolet circular dichroism (CD) spectra and the changes were inhibited in the presence of EGTA. These results identify important interactions between calcium and pCDase, which may play an essential role in the interaction of pCDase and its substrate.

Discovery of synthetic penaeidin activity against antibiotic-resistant fungi.

Penaeidins are antimicrobial peptides from shrimp that are constituted by divergent classes of peptide isoforms in an individual organism. Penaeidin sequence variation suggests functional diversity in the host and promises differential activities if applied to treat infections in humans. We have synthesized isoform 4 of penaeidin class 3 from the Atlantic shrimp, Litopenaeus setiferus, by native ligation using three peptide segments. Our synthesis approach led to the discovery of an irreversible side reaction that was successfully suppressed, a discovery, which has particular relevance to the synthesis of cysteine-rich peptides. The antimicrobial activity of full-length penaeidin and the N-terminal proline-rich domain of this isoform were compared with the corresponding peptides of penaeidin class 4 isoform 1 using a wide range of bacteria and fungi. New aspects of penaeidin function are reported that include activity against fungi of the phylum Basidiomycota (Cryptococcus strains), activity against fungi that are pathogenic to humans and effectiveness in the context of antibiotic resistance mechanisms (Cryptococcus and Candida spp.). The proline-rich domain of penaeidin class 4 shows the highest relative antimicrobial activity, while exhibiting no cytotoxicity to human monocytes, and therefore stands out as a potential peptide therapeutic.

The mode of interaction of the relaxin-like factor (RLF) with the leucine-rich repeat G protein-activated receptor 8.

The relaxin-like factor (RLF, also named INSL3) is a critical component in the chain of events that lead to the normal positioning of the gonads in the male fetus. RLF and relaxin share features of the secondary structure to the extent that relaxin cross-reacts with the LGR8, the RLF receptor. Although both hormones interact with their receptors essentially via the B chain, the sharply defined binding cassette of relaxin is not present in RLF. Structure and function analysis of RLF derivatives with single amino acid replacements revealed that the most important binding residues are tryptophan B27, followed by arginine B16 and valine B19. Single alanine replacements for each individual position resulted in a relative receptor affinity of 4.0% (B16), 6.1% (B19), and 0.5% (B27). Tryptophan B27 is located on an extended structure, and arginine B16 and valine B19 are positioned on the exposed surface of the B chain helix. The 3 residues could be brought together to form a contiguous binding area if the C-terminal end of the B chain were free to fold back against the central portion of the B chain helix. Such a movement depends critically on the flexibility of the C-terminal end, which is controlled by positions B23-25. In as much as these major binding residues seem hardly sufficient to explain the strong binding of RLF to LGR8 we searched for and found an extended region where little contributions by individual residues added up to a strong receptor affinity. This mode of interaction could drive the binding energy sufficiently high to account for the picomolar binding constant of RLF and its receptor.

Human decidual relaxin and preterm birth.

Relaxin in human pregnancy is both a systemic hormone from the corpus luteum and an autocrine/paracrine hormone at the maternal-fetal interface formed by the decidua/placenta and fetal membranes. We have focused our studies on the autocrine/paracrine roles of relaxin, especially in the preterm premature rupture of the fetal membranes, which causes 30-40% of preterm births. By using different techniques and different tissue collections, our laboratory has shown that expression of the relaxin genes and proteins in the decidua and placenta is increased in patients with preterm premature rupture of the fetal membranes. Relaxin binding and the expression of LGR7 are primarily in the chorion and decidua and are downregulated after spontaneous labor and delivery both at term and preterm. However, expression of LGR7 in the fetal membranes is significantly greater in all clinical situations at preterm than term, suggesting an important role for relaxin in these tissues at that time. The roles of the relaxin system in three potential causes of preterm birth are discussed: in the growth and proliferation of the membranes important for fetal membrane accommodation to fetal and placental growth, in acute infection, and in the inflammatory response leading to the initiation of labor.

LGR8 signal activation by the relaxin-like factor.

The relaxin-like factor (RLF) is thought to be responsible for the intra-abdominal migration of the testis during mammalian development. Our latest studies of RLF and LGR8 have revealed that the N-terminal region of the A chain is not required for receptor binding but is indispensable for cyclic AMP generation. RLF derivatives with six residues deleted from the N terminus of the A chain are active, whereas further truncation, up to the first A chain cysteine (A-10), yields tightly binding ligands devoid of signaling activity. These derivatives are specific competitive inhibitors (RLFi) of RLF. Although receptor binding is dependent upon B chain residues, the N-terminal region of the A chain is a generic trigger of the trans-membrane signaling activity.

Solution structure of synthetic penaeidin-4 with structural and functional comparisons with penaeidin-3.

Antimicrobial peptide structure has direct implications for the complexity of functions and mechanisms of action. The penaeidin antimicrobial peptide family from shrimp is divided into multiple class designations based on primary structure. The penaeidin classes are not only characterized by variability in primary sequence but also by variation in target specificity and effectiveness. Whereas class 4 exhibits low isoform diversity within species and is highly conserved between species, the primary sequence of penaeidin class 3 is less conserved between species and exhibits considerable isoform diversity within species. All penaeidins, regardless of class or species, are composed of two dramatically different domains: an unconstrained proline-rich domain and a disulfide bond-stabilized cysteine-rich domain. The proline-rich domain varies in length and is generally less conserved, whereas the spacing and specific residue content of the cysteine-rich domain is more conserved. The structure of the synthetic penaeidin class 4 (PEN4-1) from Litopenaeus setiferus was analyzed using several approaches, including chemical mapping of disulfide bonds, circular dichroism analysis of secondary structural characteristics, and complete characterization of the solution structure of the peptide by proton NMR. L. setiferus PEN4-1 was then compared with the previously characterized structure of penaeidin class 3 from Litopenaeus vannamei. Moreover, the specificity of these antimicrobial peptides was examined through direct comparison of activity against a panel of microbes. The penaeidin classes differ in microbial target specificity, which correlates to variability in specific domain sequence. However, the tertiary structure of the cysteine-rich domain and indeed the overall structure of penaeidins are conserved across classes.

The trap-like relaxin-binding site of the leucine-rich G-protein-coupled receptor 7.

The pleated sheet region of the leucine-rich G-protein-coupled receptor 7 supports a relaxin-binding group of amino acids that perfectly matches the binding cassette of relaxin. Arginines B13 and B17 are each chelated by an aspartic acid/glutamic acid pair and by isoleucine B20, which, offset by a one-quarter helix turn from a straight line connecting the arginines, interacts with a cluster of hydrophobic amino acids. The binding cassette of relaxin cuts at an angle of approximately 45 degrees across five parallel leucine-rich repeats. The arginine residues 13 and 17, which evolve parallel from the B-chain alpha-helix of relaxin, neutralize the charge repulsion of the juxta-posed acidic groups on the receptor and thereby trigger closure of a hydrogen bonding network around the guanidinium groups. Thus, relaxin is bound by synchronized chelation of two arginines and stabilized by hydrophobic interaction of isoleucine B20 with tryptophan, isoleucine, and leucine in neighboring leucine-rich repeats of the receptor. Deletion of any one of the three features diminishes interaction to the level of nonspecific binding. This model explains the exquisite sensitivity of relaxin binding avidity to minute changes in the disposition of the guanidinium and the size dependence of the hydrophobic binding residue in position B20.

Synthetic cross-links arrest the C-terminal region of the relaxin-like factor in an active conformation.

All kinetic indicators suggest that the receptor-binding site of the relaxin-like factor (RLF) is located in the flexible C-terminal region of the B chain and is centered about the tryptophan in position B27. Conformational restraints of varying lengths were used to confine Trp (B27) to a narrow range of positions relative to the C terminus of the A chain. Total synthesis of variants involving residues proximate to Trp (B27) assured us that none had a role in receptor binding. Even arginine B26, next to the important tryptophan, can be replaced without deleterious effects. To fix the distance between the C-terminal end of the A chain and Trp (B27) at predetermined lengths, we synthesized RLF with covalent cross-links between a lysine, which was placed in position B26, and the alpha-carboxyl group at the C terminus of the A chain (A26). The affinity of the cross-linked ligands for the RLF receptor varied as a parabolic function of length whereby the range of 10.0-11.1 A provided the closest approach to the binding conformation. Wild-type transmembrane signaling activity, as determined by cAMP accumulation, was reached only with a glycine cross-linker.

A new class (penaeidin class 4) of antimicrobial peptides from the Atlantic white shrimp (Litopenaeus setiferus) exhibits target specificity and an independent proline-rich-domain function.

A highly pure, chemically defined representative of a new class of antimicrobial peptide from the Atlantic white shrimp (Litopenaeus setiferus), penaeidin class 4 [Pen4-1 (penaeidin class 4 isoform 1)], was produced synthetically. Chemical synthesis was achieved by native ligation from two separate domains yielding a bioactive peptide that reflected the characteristics of native penaeidin. Synthetic Pen4-1 proved to be an effective antimicrobial peptide, particularly against the broad-spectrum pathogen Fusarium oxysporum, exhibiting a complex effect on reproductive growth at inhibitory concentrations resulting in the suppression of spore formation. Pen4-1 exhibits unique features [not previously observed for penaeidins from the Pacific white shrimp (L. vannamei)], including target-species specificity against Gram-positive bacteria, indicating a potential partitioning of antimicrobial function among this family of peptides. The proline-rich domain of penaeidin class 4 alone was an active antimicrobial peptide, having the same target range as the full-length Pen4-1. These findings indicate that the proline-rich domain of penaeidin is sufficient to confer target specificity and that divergence in this domain between classes can result in a gain in antimicrobial function as observed for the proline-rich domain of Pen4-1.

Abnormal relaxin secretion during pregnancy in women with type 1 diabetes.

To test the hypothesis that relaxin may play a role in the fetal abnormalities associated with pregnancy in type 1 diabetic women, we previously compared gestational relaxin concentrations in diabetic and clinically normal women using a porcine relaxin radioimmunoassay (RIA): Serum immunoactive relaxin was significantly (P < 0.001) elevated in the diabetic women. To confirm and extend this work in a larger group of subjects, we have now used an enzyme-linked immunosorbent assay (ELISA) specific for human H2 relaxin (the normal human gene product) to determine immunoactive serum relaxin concentrations in serial samples from 61 Type 1 diabetic and 21 normal pregnant women. Samples from 22 of the diabetic and nine of the normal women were also directly compared in the porcine relaxin RIA. ELISA-determined serum relaxin was higher (P < 0.001) at 24 and 36 weeks of pregnancy in type 1 diabetic women than in controls, confirming previous findings. However, the geometric mean increase in immunoactive relaxin concentration in identical samples from pregnant diabetic women over that of controls was significantly greater with the RIA than with the ELISA (271% vs 44%; P < 0.001). To investigate this discrepancy, the specificity and epitope selectivity of the RIA and the ELISA were compared using several synthetic polypeptides, including human relaxins H1 and H2, and relaxin and insulin derivatives. Both assays showed great specificity, but the porcine RIA selectively identified the epitopes of the receptor-binding domain of the relaxin B chain and cross-reacted strongly with H1 and H2 relaxins. In contrast, only the H2 peptide was detected by the ELISA antiserum. Therefore, the marked discrepancy between the RIA and the ELISA could be due to the presence in the diabetic samples of another relaxin-like molecule in addition to the normal H2 relaxin. The biological consequences of elevated serum relaxin in diabetic pregnancy remain to be elucidated.