Short carboxyl terminal parathyroid hormone peptides modulate human parathyroid hormone signaling in mouse osteoblasts.

BACKGROUND: Novel human parathyroid hormone (hPTH) peptides of unknown biological activity have recently been identified in the serum of subjects with normal renal function, chronic renal failure, and end-stage renal disease through the application of liquid chromatography-high resolution mass spectrometry. PURPOSE: of experiments: To determine the bioactivity of these peptides, we synthesized hPTH28-84, hPTH38-84, and hPTH45-84 peptides by solid phase peptide synthesis and tested their bioactivity in MC3T3-E1 mouse osteoblasts, either individually or together with the native hormone, hPTH1-84, by assessing the accumulation of 3´,5´-cyclic adenosine monophosphate (cAMP) and the induction of alkaline phosphatase activity. RESULTS: Increasing doses of hPTH1-84 (1-100 nM) increased the accumulation of cAMP and alkaline phosphatase activity in osteoblasts. hPTH28-84, hPTH38-84, and hPTH45-84 in concentrations of 1-100 nM were biologically inert. Surprisingly, 100 nM hPTH38-84 and hPTH45-84 increased the accumulation of cAMP in osteoblasts treated with increasing amounts of hPTH1-84. Human PTH28-84 had no effects on cAMP activity alone or in combination with hPTH1-84. Conversely, 100 nM hPTH38-84, hPTH45-84, and hPTH28-84 blocked the activation of alkaline phosphatase activity by hPTH1-84. CONCLUSIONS: The data show that the short carboxyl-terminal hPTH peptides, hPTH38-84 and hPTH45-84, increase the amount of cellular cAMP generated in cultured osteoblasts in response to treatment with full-length hPTH1-84 when compared to full-length hPTH1-84 alone. Human PTH28-84 had no effect on cAMP activity alone or in combination with hPTH1-84. Human PTH28-84, hPTH38-84 and hPTH45-84 reduced the effects of hPTH1-84 in osteoblasts with respect to the induction of alkaline phosphatase activity compared to hPTH1-84 alone. Short carboxyl peptides of human PTH are biologically inert but when administered together with full-length hPTH1-84 modulate the bioactivity of hPTH1-84 in osteoblasts.

Prospects in the total synthesis of protein therapeutics.

Protein therapeutics are gaining headway in the treatment and prevention of a variety of maladies. Critical to the success and growing use of biologic-level pharmaceuticals is the development of methods for their precise synthesis. This review highlights the emerging techniques for the total synthesis of proteins, with a special focus on the possibility of accessing therapeutic targets through chemoselective peptide ligations.

Engineering of therapeutic polypeptides through chemical synthesis: early lessons from human parathyroid hormone and analogues.

Application of chemical synthesis to gain access to high purity hPTH as well as more stable analogues was accomplished through a menu of extended NCL followed by metal free dethiylation.

Application of the logic of cysteine-free native chemical ligation to the synthesis of Human Parathyroid Hormone (hPTH).

The power of chemical synthesis of large cysteine-free polypeptides has been significantly enhanced through the use of nonproteogenic constructs which bear strategically placed thiol groups, enabling native chemical ligation. Central to these much expanded capabilities is the specific, radical-induced, metal-free dethiolation, which can be accomplished in aqueous medium.

Structure-function studies of analogues of parathyroid hormone (PTH)-1-34 containing beta-amino acid residues in positions 11-13.

The 1-34 N-terminal fragments of human parathyroid hormone (PTH) and PTH-related protein (PTHrP) elicit the full spectrum of bone-relevant activities characteristic of the intact hormones. The structural elements believed to be required for receptor binding and biological activity are two helical segments, one N-terminal and one C-terminal, connected by hinges or flexible points located around positions 12 and 19. To test this hypothesis, we synthesized and characterized the following analogues of PTH-(1-34), each containing single or double substitutions with beta-amino acid residues around the putative hinge located at position 12: I. [Nle(8,18),beta-Ala(11,12),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); II. [Nle(8,18),beta-Ala(12,13),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); III. [Nle(8,18),beta-Ala(11),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); IV. [Nle(8,18),beta-hLeu(11),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); V. [Nle(8,18),beta-Ala(12), Nal(23),Tyr(34)]bPTH-(1-34)NH(2); VI. [Nle(8,18),beta-Ala(13), Nal(23),Tyr(34)]bPTH-(1-34)NH(2) (beta-hLeu = beta-homo-leucine; beta-Ala = beta-alanine; Nal = L-2-naphthyl-alanine; Nle = norleucine). Analogues I and III exhibit very low binding affinity and are devoid of adenylyl cyclase activity. Analogue II, despite its very low binding capacity is an agonist. Biological activity and binding capacity are partially restored in analogue IV, and completely restored in analogues V and VI. The conformational properties of the analogues were investigated in aqueous solution containing dodecylphosphocholine (DPC) micelles as a membrane-mimetic environment using CD, 2D-NMR, and molecular dynamics calculations. All peptides fold partially into the alpha-helical conformation in the presence of DPC micelles, with a maximum helix content in the range of 30-35%. NMR analysis reveals the presence of two helical segments, one N-terminal and one C-terminal, as a common structural motif in all analogues. Incorporation of beta-Ala dyads at positions 11,12 and 12,13 in analogues I and II, respectively, enhances the conformational disorder in this portion of the sequence but also destabilizes the N-terminal helix. This could be one of the possible reasons for the lack of biological activity in these analogues. The partial recovery of binding affinity and biological activity in analogue IV, compared to the structurally similar analogue III, is clearly the consequence of the reintroduction of Leu side-chain of the native sequence. In the fully active analogues V and VI, the helix stability at the N-terminus is further increased. Taken together, these results stress the functional importance of the conformational stability of the helical activation domain in PTH-(1-34). Contrary to expectation, insertion of a single beta-amino acid residue in positions 11, 12, or 13 in analogues III-VI does not favor a disordered structure in this portion of the sequence.

Structure and activities of constrained analogues of human parathyroid hormone and parathyroid hormone-related peptide: implications for receptor-activating conformations of the hormones.

Parathyroid hormone (PTH) has a helix-bend-helix structure in solution. Part of the C-terminal helix, residues 21-31, is amphiphilic and forms a critical receptor-binding region. Stabilization of this alpha-helix by lactam formation between residues spaced i, i + 4 on the polar face was previously reported to increase adenylyl cyclase-stimulating (AC) activity if between residues 22 and 26 but to diminish it if between residues 26 and 30 [Barbier et al. (1997) J. Med. Chem. 40, 1373-1380]. This work reports the effects of other cyclizations on the polar face, differing in ring size or position, on alpha-helix conformation, as measured by circular dichroism, and on AC-stimulating activity. All analogues cyclized between residues 22 and 26 had at least a 1. 5-fold increase in activity, suggesting an alpha-helical structure between about residues 21 and 26. Cyclization between residues 25 and 29 or residues 26 and 30 diminished activity by 20-30%, despite stabilizing alpha-helix, suggesting that residues 25-31 bind to the receptor in a helical, but not classical alpha-helical, conformation. Analogues cyclized between residues 13 and 17 had slightly increased activity. A bicyclic analogue, with lactams between residues 13 and 17 and residues 22 and 26, had about the same activity as that cyclized only between 22 and 26. Parathyroid hormone-related peptide (PTHrP) may bind in a manner similar to the common receptor, but hydrophobic moment calculations suggest that it must bind as a tighter helix in order to optimally present its hydrophobic residues to the receptor. Both PTHrP analogues cyclized between either residues 22 and 26 or residues 26 and 30 had more stable alpha-helices but reduced AC activities, consistent with this hypothesis.

Design and applications of parathyroid hormone analogues.

The endocrine parathyroid hormone (PTH) is the major regulator of serum calcium levels. In contrast, the autocrine/paracrine parathyroid hormone-related peptide (PTHrP) has been associated with organism development. Both are secreted as much larger molecules but have their major functions associated with their N-terminal 34 residues. They share a common receptor expressed in organs critical to PTH function - bone, kidney, and intestine. PTH and PTHrP receptor activation stimulates adenylyl cyclase (AC), phospholipase C (PLC), and phospholipase D (PLD) in target cells. It has been possible to separate the AC-stimulation from that of PLC. AC-stimulation requires at least the N-terminal 28 residues of PTH and PLC-stimulation requires a minimum of residues 29-32-NH2. Intermittent administration of PTH stimulates bone growth and requires AC-stimulation. The shortest linear sequence of hPTH with essentially full anabolic activity for bone growth-stimulation is hPTH(1-31)NH2. Two applications are postulated for PTH and PTHrP-based pharmaceuticals - treatment of bone loss due to osteoporosis and reversal of the hypercalcemic effect of malignancy. PTHrP analogues which strongly inhibit PTHrP AC-stimulation showed promise for the treatment of malignancy-associated hypercalcemia in animal trials but failed in human ones. However, both animal and human trials of hPTH have shown significant bone growth-stimulating effects. New deletion, substitution and cyclized analogues of PTH show great promise both for greater in vitro activity and possibly for improved delivery and greater specificity as agents for restoration of bone loss in osteoporosis.

Probing the bimolecular interactions of parathyroid hormone with the human parathyroid hormone/parathyroid hormone-related protein receptor. 1. Design, synthesis and characterization of photoreactive benzophenone-containing analogs of parathyroid hormone.

Parathyroid hormone (PTH) regulates calcium and phosphate metabolism through a G-protein-coupled receptor which is shared with PTH-related protein (PTHrP). Therefore, structure-activity studies of PTH and PTHrP with their common receptor provide an unusual opportunity to examine the structural elements in the two hormones and their common receptor which are involved in the expression of biological activity. Our approach to studying the nature of the bimolecular interface between hormone and receptor is to use a series of specially designed photoreactive benzophenone- (BP-) containing PTH analogs in "photoaffinity scanning" of the PTH/PTHrP receptor. In this report we describe a series of BP-containing agonists and antagonists which have been synthesized by solid-phase methodology and characterized physiocochemically and biologically. Each of the 12 analogs contains a single BP moiety at a different defined position. Examples of BP-containing agonists prepared and studied in human osteogenic sarcoma Saos-2/B-10 cells are [Nle8,18,Lys13(epsilon-pBZ2),L-2-Nal23,Tyr34]bPTH(1-34 )NH2(K13)(Kb = 13 nM; Km = 2.7 nM) and [Nle8,18,L-Bpa23,Tyr34[bPTH(1-34)NH2(L-Bpa23) (Kb = 42 nM; Km = 8.5 nM). Another BP-containing analog, [Nle8,18,D-2-Nal12,Lys13(epsilon-pBZ2),L-2-Nal23 ,Tyr34]bPTH(7-34)NH2, was a potent antagonist (Kb = 95 nM; Ki = 72 nM). The amino acids substituted by residues carrying the BP moiety span the biologically active domain of the hormone (Phe7, Gly12, Lys13, Trp23, and Lys26). Analysis of photo-cross-linked conjugates of PTH/PTHrP receptor with BP-containing PTH analogs should help to identify the "contact points" between ligand and receptor.

Structure of human parathyroid hormone 1-37 in solution.

Human parathyroid hormone (hPTH), amino acids Ser1 to Leu37, is biologically active with respect to both receptor binding and activation of adenylate cyclase to influence the serum calcium concentration. It induces DNA synthesis via an unknown signal pathway. We investigated the structure of hPTH(1-37) in H2O/buffer solution under near physiological conditions, that is pH 6.0 and 270 mM salt, by circular dichroism, ultracentrifugation, nuclear magnetic resonance spectroscopy, and molecular dynamics calculations. Complete sequence specific assignments of all 1H resonances were performed by using 1H two-dimensional NMR measurements (double quantum-filtered correlated spectroscopy, nuclear Overhauser effect spectroscopy (NOESY), and total correlation spectroscopy with suppression of NOESY-type cross-peaks spectra). hPTH(1-37) obtained helical structure and showed hydrophobic interactions defining a tertiary structure. The NH2-terminal four amino acids of hPTH(1-37) did not show a stable conformation. Evidence for an alpha-helical region between Ile5 and Asn10 was found. This region was followed by a flexible link (Gly12, Lys13) and a well defined turn region, His14 to Ser17. The latter was stabilized by hydrophobic interactions between Trp23 and Leu15. Ser17 through at least Leu28 formed an alpha-helix. Arg20 and Lys27 were involved in the core built by His14 to Ser17. Unrestrained molecular dynamics simulations indicated that the structure was stable on the 200 ps time scale.

Comparison of the pharmacokinetics of parenteral parathyroid hormone-(1-34) [PTH-(1-34)] and PTH-related peptide-(1-34) in healthy young humans.

The amino-terminal fragments of human PTH [hPTH-(1-34)] and PTH-related peptide [PTHrP-(1-34)] appear to be equipotent in several rodent models. However, continuous i.v. infusions of these peptides to young human volunteers suggested that a 10-fold higher molar dose of PTHrP was required to produce comparable circulating levels of the peptide and biochemical responses similar to PTH. As PTHrP has a wide variety of target tissues in mammalian species and may, therefore, play a paracrine, rather than an endocrine, hormonal role in vivo, we evaluated whether enhanced metabolic clearance of injected PTHrP might explain its apparently reduced potency as a PTH-like hormone. Ten healthy subjects [age, 25 +/- 9 (+/- SD) yr] received in random order either hPTH-(1-34) or hPTHrP-(1-34) given by bolus i.v. injections in a dose of 10.7 nmol. Measurements of plasma immunoreactive peptide indicated a comparable volume of distribution for each, but the apparent t1/2 (8.3 +/- 1.6 min) and plasma clearance (4.0 +/- 1.4 L/min) for hPTHrP were significantly (P < 0.05) accelerated compared to those of hPTH (t1/2, 10.2 +/- 0.5 min; clearance, 2.0 +/- 0.4 L/min). Peak plasma cAMP levels were 9-fold lower in response to hPTHrP (29.5 +/- 19 vs. 190 +/- 63 pmol/L; P < 0.01), and increases in urinary cAMP excretion were 5-fold lower (2.1 +/- 1.1 vs. 11.2 +/- 3.7 nmol/mmol creatinine; P < 0.01). No major differences were observed in the urinary excretion of phosphate, calcium, or sodium between the two peptides. Although hPTHrP-(1-34) has a 2-fold higher MCR than hPTH-(1-34), this may not explain the more than 5-fold lower plasma or urinary cAMP response to PTHrP in humans. The comparable effects of PTH and PTHrP on urinary phosphate, calcium, and sodium may indicate a non-cAMP-dependent pathway for these responses, although the intracellular pool of cAMP generated to either peptide, and thus the local target tissue response, could not be estimated in the present study.

Structure and protein kinase C stimulating activities of lactam analogues of human parathyroid hormone fragment.

Five analogues of human parathyroid hormone (hPTH-(20-34)-NH2, I; cyclo[Lys26-Asp30]-hPTH-(20-34)-NH2, II; cyclo[Glu22-Lys26]-hPTH-(20-34)-NH2, III; cyclo[Lys27-Asp30]- hPTH-(20-34)-NH2, IV; and [Leu27]-hPTH-(20-34)-NH2 V) were tested for their ability to promote membrane-bound protein kinase C (PKC) activity in a rat osteosarcoma cell line (ROS 17/2). Analogues I, II and V stimulated PKC activity in the picomolar range, whereas analogues III and IV did not stimulate this activity at any concentration tested. The circular dichroism spectra in neutral, aqueous buffer showed an increase in alpha-helix in analogues II, III and V as compared to I; this increase appeared to be in the region of the cyclic lactam structure. Analogue IV did not adopt a helical structure, even in the presence of 40% trifluoroethanol, a helix-promoting solvent. The remaining analogues showed a three- to four-fold enhancement of alpha-helix in this solvent. Analogues II and III had increased retention times in reversed-phase chromatography, as compared to I and IV. This is consistent with a stabilization of amphiphilic helix in analogues II and III compared with I and IV. The data suggest that in the region bounded approximately by residues 24-32, an amphiphilic alpha-helix is important for correct functional binding to the PTH receptor.

Stabilized NMR structure of human parathyroid hormone(1-34).

The structure of the biologically-active N-terminal region of human parathyroid hormone, PTH(1-34), was investigated in the presence of 10% trifluoroethanol using two-dimensional proton NMR spectroscopy, distance geometry and dynamic simulated annealing. Complete assignments of all backbone and side chain hydrogens were made with the aid of totally correlated spectroscopy (TOCSY) experiments, providing through-bond 1H-1H connectivities, and NOESY providing through-space and sequential backbone connectivities. Distance and angle constraints were used in the distance geometry algorithm DIANA II to generate a family of structures satisfying all inputs. The lowest energy structures were further refined using the dynamic-simulated-annealing protocol XPLOR 3. The major structural features evident in 10% trifluoroethanol are two segments of alpha-helix extending from residues Glu4 to Lys14/His14 and Ser17 to Asp30. A short length of unordered structure joined the two spans of helix. The structures of the N-terminal regions (4-13/14) agreed closely with the structure found in human parathyroid-hormone-related protein (PTHrP)(1-34) obtained earlier by Ray, Barden and Kemp [19]. However, PTHrP(16-19) exhibited a reverse turn which is incorporated in an extended helix in PTH. The consequent interactions between several hydrophobic residues in the C-terminal region in PTHrP are absent in PTH. Moreover, the turn in PTHrP(22-25) at the start of the C-terminal helix is present as a more standard loop of helix in PTH. Comparisons between the structures of the two hormones has enabled the probable location and structure of the PTH receptor-binding site to be placed in the segment of amphiphilic alpha-helix (24-31).

FTIR spectroscopy study of PTHrP(1-34) involved in humoral hypercalcaemia of malignancy.

The components of secondary structure of the biologically-active N-terminal domain of human parathyroid-hormone-related protein (residues 1-34) and several truncated species were examined using Fourier transform infrared (FTIR) spectroscopy. The major structural features include a segment of alpha-helix within the N-terminal segment probably extending from Glu-4 to Lys-11 with three beta-turns localized to the segments Gly-12 to Ile-15, Gln-16 to Arg-20 and His-25 to Ala-29. Some beta-sheet was detected in the full-length peptide, but not in any of the C-terminal truncated samples. These structural features were studied in the smaller peptides for the purpose of localization of the various components and with a view to describing the region likely to form the bulk of the receptor binding site.

Synthesis of fully active biotinylated analogues of parathyroid hormone and parathyroid hormone-related protein as tools for the characterization of parathyroid hormone receptors.

The synthesis, purification, and characterization of biotinylated analogues of parathyroid hormone (PTH) and PTH-related protein (PTHrP) are described. A novel methodology was developed which allowed the selective biotinylation during solid-phase synthesis of either the Lys13 or Lys26 residue in PTH/PTHrP sequences. Incorporation of orthogonally protected N alpha-Boc-Lys(N epsilon-Fmoc) at a selected position in the sequence, followed by selective side-chain deprotection and biotinylation of the epsilon-amino group, permitted modification of the specific lysine only. Biotinylated analogues of [Nle8,18,Tyr34]bPTH(1-34)NH2 (analogue 1a) were prepared by modification of Lys13 with a biotinyl group (analogue 1) or a biotinyl-epsilon-aminohexanoyl group (analogue 2) or at Lys26 with a biotinyl-epsilon-aminohexanoyl group (analogue 3). A biotinylated PTHrP antagonist [Leu11,D-Trp12,Lys13(N epsilon-(biotinyl-beta-Ala))]PTHrP(7-34)NH2 (analogue 5), was also prepared. In a different synthetic approach, selective modification of the thiol group of [Cys35]PTHrP(1-35)NH2, in solution, with N-biotinyl-N'-(6-maleimidohexanoyl)hydrazide, resulted in analogue 4. The high affinities of the biotinylated analogues for PTH receptors present in human osteosarcoma B-10 cells or in porcine renal cortical membranes (PRCM), were comparable to those of the underivatized parent peptides. The analogues were also highly potent in stimulation of cAMP formation (analogues 1-4) or inhibition of PTH-stimulated adenylyl cyclase (analogue 5) in B-10 cells. The most potent analogue (analogue 1) had potencies in B-10 cells (Kb = 1.5 nM, Km = 0.35 nM) and in porcine renal membranes (Kb = 0.70 nM) identical or similar to those of its parent peptide, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Solid-phase synthesis and biologic activity of human parathyroid hormone (1-84).

We have chemically synthesized the full-length, 84 amino acid, human parathyroid hormone (hPTH) on a greater than 100 mg scale by the Merrifield solid-phase technique of stepwise peptide synthesis using a benzhydrylamine support. The peptide was purified by high-performance liquid chromatography and found to be greater than 96% pure. The authenticity or the sequence of the synthetic peptide was confirmed by repetitive Edman degradation. Furthermore, tryptic digestion of hPTH generated the predicted fragments. The synthetic full-length hormone was evaluated for biologic activity in assays of PTH receptor binding and stimulation of adenylate cyclase activity (using bovine renal cortical membranes and rat and human bone cells). Synthetic hPTH (1-84) was found to be highly potent in binding to PTH receptors (Kb = 1-25 nM) and stimulating adenylate cyclase (Km = 1-14 nM). The availability of significant quantities of synthetic full-length hPTH and future analogs will permit widespread use in multiple in vitro and in vivo assays to delineate their spectrum of biologic properties. Available supplies of the synthetic hormone will also enable evaluation of the effectiveness of PTH antagonists at inhibiting the action of native sequence hormone at its receptors.

Biological properties of synthetic human parathyroid hormone: effect of deamidation at position 76 on agonist and antagonist activity.

Recent studies have suggested a role for the carboxyl-terminus of PTH in the binding of the molecule to renal and skeletal receptors, but the functional significance of this binding remains uncertain. We have investigated the possible role of this region by examining the effect of substituting the asparagine residue at position 76 of the native human molecule [Asn76]hPTH-(1-84) with an aspartate residue, [Asp76] hPTH-(1-84) on activity in both renal and skeletal cytochemical (CBA) and adenylate cyclase (AC) bioassays. In the renal CBA, [Asp76]hPTH-(1-84) was considerably less potent than [Asn76]hPTH-(1-84) and produced dose-dependent inhibition of the bioactivity of intact bovine (b) PTH-(1-84), bPTH-(1-34), and [Asn76]hPTH-(1-84). [Asp76]hPTH-(39-84) inhibited the response to intact PTH to a lesser extent, whereas [Asp76]hPTH-(53-84) had no antagonistic activity. In the metatarsal CBA, [Asp76]hPTH-(1-84) inhibited the response to intact PTH, but was less potent than in the renal CBA. In both renal (OK) and skeletal (UMR) cell AC assays [Asp76]hPTH-(1-84) and [Asn76]hPTH-(1-84) were equipotent agonists. Therefore, the CBAs are much more sensitive to modification of the carboxyl end of the molecule than AC assays. The antagonist properties of [Asp76]hPTH-(1-84) appeared to be mediated by phosphodiesterase activation as theophylline abolished the antagonism of this analog. These studies indicate that generation of PTH analogs, modified at the carboxyl-terminal region as well as at the amino-terminus, may be useful for developing potent PTH antagonists.

Biological activity of parathyroid hormone antagonists substituted at position 13.

Lysine occupies position 13 in the parathyroid hormone (PTH) antagonist, [Nle8,18,Tyr34]bPTH(7-34)NH2. Acylation of the epsilon-amino group in lysine 13 by a hydrophobic moiety is well tolerated in terms of bioactivity: the analog [Nle8,18, D-Trp12,Lys 13 (epsilon-3-phenylpropanoyl),Tyr34]bPTH(7-34)NH2 is equivalent to the parent peptide in its affinity for PTH receptors and its ability to inhibit PTH-stimulated adenylate cyclase in both kidney- and bone-based assays. Truncation of this peptide by deletion of phenylalanyl7 with concomitant removal of the amino-terminal alpha-amino group yielded the analog desamino[Nle8,18,D-Trp12,Lys13 (epsilon-3-phenylpropanoyl),Tyr34]bPTH(8-34)NH2, an antagonist of high potency in vitro (Kb = 4 and 9 nM, Ki = 73 and 3.5 nM in kidney- and bone-based assays, respectively). Also this analog is potentially stable to aminopeptidases present in many biological systems.

Preparation and characterization of [N alpha-(4-azido-2-nitrophenyl)Ala1,Tyr36]-parathyroid hormone related peptide (1-36)amide: a high-affinity, partial agonist having high cross-linking efficiency with its receptor on ROS 17/2.8 cells.

The synthesis, purification, and structural analysis of the major compounds resulting from photoderivatization of [Tyr36]-parathyroid hormone related peptide (1-36)amide [[Tyr36]PTHrP(1-36)amide] are described. The reaction of the synthetic peptide with 4-fluoro-3-nitrophenyl azide under nonaqueous conditions yields three major products (peaks D-1, D-2, and G), which were purified to homogeneity by reverse-phase high-performance liquid chromatography. Subsequent amino acid analysis showed that the peptides of peaks D-1 and G each lack one lysine residue, while the peptide in peak D-2 lacks one alanine residue, suggesting that these residues are chemically modified by photoderivatization. Sequence analysis of the photoderivatized peptides revealed that compounds D-1 and G were derivatized on Lys13 and Lys11, respectively. Compound D-2 was N-blocked, indicating that this compound is derivatized on the alpha-amino function of Ala1. Both Lys residues of D-2 were quantitatively recovered upon sequencing after digestion with endoproteinase Glu-C. Compounds D-2 and G had apparent KdS of 1 X 10(-9) M and 0.6 X 10(-9) M, respectively, for their receptors on ROS 17/2.8 cells, which are identical with or similar to that of the underivatized [Tyr36]PTHrP(1-36)amide. Compound G had the same adenylate cyclase stimulating potency as the underivatized, synthetic [Tyr36]PTHrP(1-36)amide, whereas compound D-2 was only a partial agonist, having about 25% of the maximal cAMP production. Compound D-1, which is modified on Lys13, retained only 2-4% of its receptor binding affinity and biological activity relative to that of its parent compound.(ABSTRACT TRUNCATED AT 250 WORDS)

Biotinylated parathyroid hormone as a probe for the parathyroid hormone receptor. Structure-function analysis and detection of specific binding to cultured bone cells by flow cytometry.

The synthetic bovine parathyroid hormone (PTH) analog (Nle8, Nle18, Tyr34) bovine PTH(1-34)amide (bPTH(1-34)amide) was reacted with biotinyl-epsilon aminocaproic acid-N-hydroxysuccinimide under conditions which yielded five isoforms which were fractionated by a combination of reversed phase and ion-exchange chromatography. These reaction products were analyzed by automated Edman degradation in a manner which allowed us to specify the location and number of biotin residues on picomole quantities of hormone. The ability of each of these isoforms to induce a rise in intracellular cAMP in the ROS 17/2.8 cell line allowed us to evaluate the effect on function of biotinylation at different residues. Derivatized PTH molecules which contained a single biotin at either lysine 13, lysine 26, or lysine 27 possessed full biological activity. However, bioactivity was significantly reduced when position 13 plus either lysine 26 or 27 were biotinylated. Biological activity was lost when all 3 lysine residues were biotinylated. Biotinylation of the alpha-NH2 group of alanine at the NH2 terminus also resulted in a total loss of activity. Hence, unlike the effect of altering the alanine at position 1, modification of a single lysine residue at positions 13, 26, and 27 has a less critical effect on biological activity of the molecule. However, biotinylation of all three lysines results in a biologically inert PTH derivative and suggests that changes in isoelectric point, hydrophobicity, or tertiary structure may strongly influence hormone function. A fully bioactive-mixture of isoforms was used to detect receptors on ROS 17/2.8 cells by flow cytometry using fluorescein isothiocyanate-avidin as a fluorescent indicator. Binding to cell surface receptors was saturable and could be inhibited by native bPTH(1-34) but not by transforming growth factor beta, calcitonin or insulin. Moreover, PTH receptors could also be detected on primary cultures of human bone cells and human fibroblasts.

Synthetic substrate for eukaryotic signal peptidase. Cleavage of a synthetic peptide analog of the precursor region of preproparathyroid hormone.

A synthetic peptide analog of the precursor region of preproparathyroid hormone has been shown to be a specific substrate for hen oviduct signal peptidase. The sequence of the 31-residue peptide is Ser-Ala-Lys-Asp-norleucine (Nle)-Val-Lys-Val-Nle-Ile-Val-Nle-Leu-Ala-Ile-Ala-Phe-Leu-Ala-Arg-Ser-As p-Gly-Lys-Ser-Val-Lys-Lys-Arg-D-Tyr-amide (Caulfield, M. P., Duong, L. T., O'Brien, R., Majzoub, J. A., and Rosenblatt, M. (1988) Mol. Endocrinol. 2, 452-458). This sulfur-free signal peptide analog can be labeled with 125I on the C-terminal D-tyrosine and is cleaved by purified hen oviduct signal peptidase between Gly and Lys, the correct site of cleavage of preproparathyroid hormone in vivo. Amino acid sequence analysis of the cleavage product released 125I at the seventh cycle of Edman degradation, confirming that enzymatic cleavage occurs at the physiological site. Synthetic peptide analogs of the substrate with Lys, Pro, or Asp substituted for Nle-18 were poor substrates for the enzyme and were also poor competitive inhibitors of catalysis, suggesting that modifications at position -18, 12 amino acids from the site of cleavage, directly influence binding by the enzyme. Analysis of the reactivity of signal peptidase with these synthetic peptides provides insight into the cleavage specificity requirements of this eukaryotic signal peptidase.