Christian Schwabe

Primary structure of the B-chain of porcine relaxin

The sequence of the B-chain of relaxin, and ovarian peptide hormone isolated from ovaries of pregnant sows, has been shown to have the following primary structure: PCA-Ser-Thr-Asn-Asp-Phe-Ile-Lys-Ala-Cys-Gly-Arg-Glu-Leu-Val-Arg-Leu-Trp-Val-Glu-Ile-Cys-Gly-Val-Trp-Ser (2820 daltons). The heterogeneity of relaxin observed during purification procedures is likely to be due to variations in the C-terminal region of the B-chain, in particular the substitution of Gln for Glu20, and the possible addition of arginine or serylarginine at the C terminus. The B-chain exhibited a distribution of sulfhydryl residues relative to one another that is identical to that found in the B-chain of insulin. A similar analogy has already been demonstrated for the A-chains of relaxin and insulin.

A fluorescent assay for proteolytic enzymes.

Abstract A method is described which permits the assay of proteolytic enzyme activity on protein substrates without precipitation or filtration steps, utilizing a fluorescent reagent which is specific for primary amines. The assay is about 100 times more sensitive than the Lowry method, much faster and less complicated. Ambiguities concerning the absorbing species are largely eliminated. The reagent (Fluorescamine, Hoffmann-La Roche RO-20-7234) yields fluorescent compounds with amino acids at pH 9.0 and with peptides at pH 6.8, but possesses no fluorescence by itself.

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 ∼45° across five parallel leucine-rich repeats. The arginine residues 13 and 17, which evolve parallel from the B-chain α-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.

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.

Relaxin: structures, functions, promises, and nonevolution.

During the last two decades synthetic chemistry and molecular biology have transformed the little‐known parturition‐mediating factor relaxin into a chemically defined entity. Relaxin is a disulfide bond analog of insulin that shows no cross‐reactivity to the insulin receptors, causes widening of the birth canal in most mammals, and has additional or different functions in various species. The receptor interaction site in relaxin has been located quite precisely in the midregion of the B chain helix and is now known to involve two arginine residues that project from the alpha helix in an n, n + 4 configuration. The A chain of relaxin which appears to be uninvolved in receptor binding, is nonetheless essential. In fact, a major structural determinant in relaxin and insulin responsible for achieving either an insulin‐like or a relaxin‐like conformation is located in the penultimate position in the intrachain loop of the A chains.—Schwabe, G. Büllesbach, E. E. Relaxin: structures, functions, promises, and nonevolution. FASEB J. 8, 1152‐1160 (1994)

The relaxin-like factor is a hormone

The relaxin-like factor (RLF) circulates in the blood-stream of humans, interacts with a membrane protein with all the characteristics of ligand-receptor binding, and must therefore be considered a hormone by definition. The polyclonal antibody raised against synthetic human RLF showed no crossreactivity to other structurally related hormones, like insulin and relaxin. The sensitivity of this assay (ED50 at 100 pM) allowed the direct measurement of RLF concentrations in serum. The highest levels were detected in the serum of postpuberty males (190 pM), whereas in females and children, the RLF concentration was one order of magnitude lower.

Specific, High Affinity Relaxin-like Factor Receptors

The relaxin-like factor (RLF) is a circulating hormone that binds to specific membrane-bound uterine receptors in the mouse. Mono-iodinated RLF tracers were produced and characterized specifically to study the properties of the RLF receptor. The tracers bound to the RLF receptor in uterine crude membrane preparations with high affinity (73 nm for 125I-Tyr(A9) RLF and 90 nm for 125I-Tyr(A26) RLF) as determined by Scatchard analysis. The specificity of binding was confirmed by chemical cross-linking experiments. Binding of 125I-Tyr(A9) RLF to the putative receptor was inhibited in the presence of a 640-fold excess of unlabeled human RLF but not by the same excess of human relaxin. SDS-gel electrophoresis of the RLF-receptor complex revealed a molecular mass of >200 kDa, which remained unchanged upon reduction. The size and the lack of subunit structure of the receptor is similar to the features reported for the relaxin receptor. In this regard both, the RLF and the relaxin receptor are different from the insulin- and the insulin-like growth factor-type 1 receptors. This observation supports the relaxin-likeness of this new factor not only toward potential target tissues but also as regards receptor features.

Demonstration of a pyroglutamyl residue at the N terminus of the B-chain of porcine relaxin.

Abstract The ovarian peptide hormone relaxin consists, like insulin, of one A- and one B-chain linked by two disulfide bonds. A peptide, isolated from a tryptic digest of the purified B-chain by high-pressure liquid chromatography (HPLC), was examined with the aid of carboxypeptidase C and a pyrrolidonecarboxylyl peptidase. In conjunction with amino acid analysis it could be demonstrated that pyrrolidonecarboxylic acid occupies the N-terminal position of a peptide with the amino acid composition Asp2, Ser, Thr, Phe, Ile, Lys. The appearance of a pyroglutamyl residue in a two-chain hormone is an interesting and unusual feature which has not yet been reported in a similar structure.

The role of RXFP2 in mediating androgen-induced inguinoscrotal testis descent in LH receptor knockout mice

LH receptor knockout (LhrKO) male mice exhibit a bilateral cryptorchidism resulting from a developmental defect in the gubernaculum during the inguinoscrotal phase of testis descent, which is corrected by testosterone replacement therapy (TRT). In vivo and in vitro experiments were conducted to investigate the roles of the androgen receptor (AR) and RXFP2 signals in regulation of gubernacular development in LhrKO animals. This study demonstrated that AR and RXFP2 proteins were expressed in the gubernaculum during the entire postnatal period. TRT normalized gubernacular RXFP2 protein levels inLhrKO mice. Organ and primary cell cultures of gubernacula showed that 5alpha-dihydrotestosterone (DHT) upregulated the expression of Rxfp2 which was abolished by the addition of an AR antagonist, flutamide. A single s.c. testosterone injection also led to a significant increase in Rxfp2 mRNA levels in a time-dependent fashion in LhrKO animals. DHT, natural and synthetic insulin-like peptide 3 (INSL3), or relaxin alone did not affect proliferation of gubernacular mesenchymal cells, while co-treatments of DHT with either INSL3 or relaxin resulted in an increase in cell proliferation, and they also enhanced the mesenchymal cell differentiation toward the myogenic pathway, which included a decrease in a mesenchymal cell marker, CD44 and the expression of troponin. These effects were attenuated by the addition of flutamide, siRNA-mediated Rxfp2 knockdown, or by an INSL3 antagonist. Co-administration of an INSL3 antagonist curtailed TRT-induced inguinoscrotal testis descent in LhrKO mice. Our findings indicate that the RXFP2 signaling pathway plays an important role in mediating androgen action to stimulate gubernaculum development during inguinoscrotal testis descent.

On the primary and tertiary structure of relaxin from the sand tiger shark (Odontaspis taurus)

The structural variability between the two known relaxin sequences from the pig [l-3] and rat [4] far exceed that observed for any insulin pair, yet their primary sequences can readily be folded into an insulin conformation [ 561. This raises the question of how much more variability in the primary sequence may be tolerated without preventing an insulin-like folding to occur and how much of the surface may be varied without loss of receptor interaction. The evolutionary diversion of relaxins from each other and the possible existence of a common ancestral gene for relaxins and insulinsled us to examine the structure of shark relaxin. Based on the assumption that sharks have existed in their present form for at least 500 million years, i.e., have lived close to the point at which fishes and the ancestors of mammals are assumed to have branched from each other and where the insulin gene might have undergone duplication to give rise to the relaxin gene, we expected to observe a closer relation between the relaxin of this quasi-prehistoric species and insulins.