Parathyroid Hormone, from Gene to Protein

The biosynthetic pathway of parathyroid hormone (PTH) has been studied from gene expression to PTH intracellular processing.' The processing of PTH has been described and involves the synthesis of an initial translational product, preProPTH, and two proteolytic cleavages that in turn produce Pro PTH and PTH. The genes and cDNAs from ten different species have been cloned, sequenced and characterized. This chapter will summarize the molecular biology of PTH, from the gene to the mRNA, the initial translational product, preProPTH and the processed mature secreted form of PTH. It will describe the sequences of the PTH gene and mRNA in different species and the specific elements in the PTH mRNA that determine mRNA processing, stability and translation. The Prepro PTH Peptide The primary form of PTH, which is stored and secreted, contains 84 amino acids. PTH is initially synthesized as a precursor, preProPTH. Two proteolytic cleavages produce the ProPTH and the secreted form of PTH. The proPTH sequence contains six extra amino acids at the N-terminus.^' Conversion of ProPTH to PTH occurrs about 15 to 20 min after biosynthesis at about the time ProPTH reached the Golgi apparatus.^ The Structure of the Pre-Peptide Evidence that the translational product of PTH mRNA was larger than ProPTH was initially obtained by translation of a crude preparation of bovine parathyroid RNA in the wheat germ cell-free system. The primary translational product migrated slower than ProPTH when analyzed by electrophoresis on either acidic-urea or sodium dodecyl sidfate-containing acrylamide gels. At that time, a similar phenomenon had been observed only for myeloma light chains.'^ In further studies, preProPTH was shown to be synthesized in cell-free systems of reticulocyte lysates.^ Translation of human parathyroid RNA also produced an analogous preProPTH.^ The observation that the carboxyl terminal peptides of bovine PTH and preProPTH were identical indicated that the extra amino acids in preProPTH were at the amino terminus. This was confirmed by incorporating selected radioactive amino acids into preProPTH and determining the location of the radioactivity by automated Edman degradation.'^ By analyzing overlap of these radioactive amino acids with those in ProPTH, the length of the bovine pre-peptide was shown to be 25 amino acids. The entire sequence of the bovine pre-peptide was determined eventually by this microsequencing technique'' and was later confirmed by Molecular Biology of the Parathyroid^ edited by Tally Naveh-Many. ©2005 Eurekah.com and Kluwer Academic / Plenum Publishers. Parathyroid Hormone, from Gene to Protein structural studies of both the bovine PTH cDNA and gene. ' The sequence of human pre-peptide was also partially determined by this microsequencing technique.^ The complete amino acid sequence was derived from the human PTH cDNA sequence ̂ ^ and later confirmed by the determination of the structure of the human gene.^ The amino acid sequence of the rat pre-peptide was derived from the sequence of the rat PTH gene^^ and partially by analysis of cloned rat PTH cDNA.^^ The amino acid sequences of the pre-pep tides show that the human and bovine pre-pep tides are 80% homologous while the rat sequence is GA% homologous to the bovine and human. ̂ This is somewhat lower than the homology of 89 and 77% in the Pro and PTH regions for bovine/human and rat/bovine-human, respectively (Fig. 1). The fact that the pre-peptide is less conserved than the rest of the molecule is consistent with pre-peptides or signal peptides of many eukaryotic proteins.^^ General structural features of the signal peptides are a central hydrophobic core and, in many cases, charged amino acids at the N-terminal and C-terminal ends of the central core. These features are largely retained in the pre-peptides of the three preProPTH molecules. Only conservative changes are present within the central core of uncharged amino acids from amino acids 10 to 21.^ Conversion ofPrePro to ProPTH The removal of the pre-peptide to produce ProPTH is mediated by an enzyme associated with microsomes. In reticidocyte and wheat germ systems that contain litde or no microsomal membranes, the primary transcriptional product of PTH mRNA is preProPTH. ' Addition of microsomal membranes from dog pancreas or chicken oviduct results in the synthesis of ProPTH.^'20 The first evidence that pre or signal peptides fiinction by binding to a limited number sites in the microsomal membrane was obtained by studies on a synthetic prePro-peptide of bovine preProPTH. The identification of the signal recognition particle as a signal peptide receptor, later on, confirmed this mechanism for most secreted and membrane proteins. The pre peptide of preProPTH is rapidly degraded after its proteolytic cleavage from preProPTH. In studies of PTH biosynthesis in intact cells, no labeled pre-peptide could be detected. The proteolytic removal of the pre-peptide probably occurs before completion of the ProPTH nascent chain, since preProPTH is difficult to detect in intact cells. Homology of the Mature PTH The mature PTH has been determined or predicted by the cDNAs in several species. The sequence of PTH of mouse, rat, man, non-human primates, horse, dog, cat, cow, pig, and chicken is shown in Figure 1. The resulting phylogenetic tree obtained from alignment of the protein sequences is shown in Figure 3A. A comparison of the amino acid sequences of PTH from several species revealed high conservation of the protein amongst all species apart from gallus (Fig. 1). In addition, three relatively conserved regions could be observed. The first two regions comprise the biologically active region of PTH and would be expected to be conserved. The addition or loss of a single amino acid at the amino terminus gready reduces biological activity, and the region is involved in binding of PTH to the receptor. In addition there is a region of conservation at the C-terminal region that is itself of interest, particularly since this region may have a separate biological effect at least on osteoclasts. Analyses of the silent changes that occur between the nucleotide sequences suggest that the conservation in the C-terminal region may be related to pre-translational events. Analysis by Perler et al described replacement changes that result in changes in amino acids and silent changes that do not alter the encoded amino acid. 10 Molecular Biology of the Parathyroid murine rat human macaca Equine canine feline bovine porcine gallus murine rat human macaca Equine canine feline bovine porcine gallus murine rat human macaca Equine canine feline bovine porcine gallus 1 MMSANTVAKV MMSASTMAKV MIPAKDMAKV MIPAKDMAKV MMSAKDMVKV MMSAKDMVKV MMSAKDMVKV MMSAKDTVKV MTSTKNLAKA 51 RMQWLRRKLQ RMQWLRKKLQ RVEWLRKKLQ RVEWLRKKLQ RVEWLRKKLQ RVEWLRKKLQ RVEWLRRKLQ RVEWLRKKLQ RVEWLRKKLQ RQDWLQMKLQ 101 GNPKS GNSKS SHEKS SHEKS SHQXS SYQKS NHQKS SHQKS SHQKS EHLRAAVQKK MIIMLAVCLL MILMLAVCLL MIVMLAICFL MIVMLAICFL MIVMFAICFL MVVMFAICFL MIVMLAICFL MVVMLAICFL IVILYAICFF DMHNFVSLGV DVHNFVSLGV DVHNFVALGA DVHNFIALGA DVHNFIALGA DVHNFVALGA DVHNFVALGA DVHNFVALGA DVHNFVALGA DVHS LGEGDKADVD LGEGDKADVD LGEADKADVN LGEADKADVD LGEADKADVD LGEADKADVD LGEADKADVD LGEADKADVD LGEADKAAVD SIDLDKAYMN pre 1 TQTDGKPVRK TQADGKPVKK TKSDGKSVKK TKSDGKSVKK K AKSDGKPVKK AKSDGKPVKK ARSDGKSVKK ARSDGKPIKK TNSDGRPMMK QMAARDGSHQ QMAAREGSYQ PLAPRDAGSQ PLAPRDAGSQ PIFHRDGGSQ PIAHRDCSSQ PIAHRDGGSQ SIAYRDGSSQ SIVHRDGGSQ ..ALEDARTQ 128 VLVKSKSQ VLVKAKSQ VLTKAKSQ VLTKAKSQ VLSKTKSQ VLTKAKSQ VLIKAKSQ VLIKAKPQ VLIKAKPQ VLFKTKPpro 1 RAVSEIQLMH RAVSEIQLMH RSVSEIQLMH RSVSEIQLMH RSVSEIQLMH RSVSEIQFMH RSVSEIQFMH RAVSEIQFMH RSVSEIQLMH RSVSEMQLMH KPTKKEENVL RPTKKEENVL RPRKKEDNVL RPRKKEDNIL RPRKKEDNVL RPLKKEDNVL RPRKKEDNVP RPRKKEDNVL RPRKKEDNVL RPRNKEDIVL 50 NLGKHLASME NLGKHLASVE NLGKHLNSME NLGKHLNSME NLGKHLNSVE NLGKHLSSME NLGKHLSSVE NLGKHLSSME NLGKHLSSLE NLGEHRHTVE 100 VD VD VE VE IE VE AE VE VE GEIRNRRLLP Figure 1. Alignment of the amino acid sequences of PTH from the 10 different species. Alignments were obtained using the default setting of PileUp program (Accelrys Inc. Madison WI). Comparison of the amino acid sequences of PTH for mouse (mus), rat, human, non human primates (macaca), horse (equine), dog (canine), cat (feline), cow (bovine), pig (porcine) and chicken (gallus). Gaps indicated by dashes were introduced to maximize the homology to the gallus sequence. The N terminal sequence of the equus PTH is not available. The arrows indicate the protolytic cleavage sites required for the conversion of preProPTH to ProPTH and PTH. The PTH mRNA Bovine preProPTH mRNA was initially more extensively characterized than the mRNAs from the other species. Preparations of bovine parathyroid RNA were obtained that contained about 50% PTH mRNA as estimated by gel electrophoresis and RNA excess hybridization to radioactive cDNA."^ The size of the mRNA was estimated to be about 750 nucleotides by sucrose gradient centrifiigation. About two thirds of the translatably active mRNA was retained by oligo(dT) cellulose, and the sizes of the poly(A) extension was broadly distributed around an average size of 60 adenylate residues, though this may be an under estimation of the actual size. While not direcdy determined, PTH mRNA probably contains a 7-methylguanosine cap since the translation of PTH mRNA was inhibited by 7-methylguanosine-5 -phosphate. The human and bovine Parathyroid Hormone, from Gene to Protein 11 PTH mRNAs appear to be heterogeneous at the 5' terminus (see section on genes). The sizes of the rat and human PTH mRNAs have been determined by Northern blot analysis to be about 800 and 850 nucleotides, respectively. ̂ '̂̂ ^ Therefore, PTH mRNAs are typical eukaryotic mRNAs that contain a 7-methyguanosine cap at the 5' terminus and a polyadenylic acid (poly A) stretch at the 3' terminus. The PTH mRNAs are twice as long as necessary to code for the primary translational product, due to 5' and 3' untranslated regions at both ends of the mRNA. Cloning of the PTH cDNAs To date the sequence of the full cDNA of rat,̂ '̂ man,^^ <iogĵ ^ cat (un published), cow,̂ ^ pig, and chicken and the partial sequence of horse^^ and non human primates^^ have been determined. The cDNA of mouse PTH was determined from the genomic PTH sequence. Table 1 shows the Gene Bank accession number for the PTH sequences of the different species and the length of the cDNAs of each of the mRNAs as they appear in the NCBI and Gene Bank databases. In addition, the hypothalamus PTH cDNA was sequenced after the PTH mRNA had been detected in neuronal tissue. ̂ '̂ The first PTH cDNAs identified were the DNAs complementary to bovine^" '̂̂ ^ and human^ ̂ PTH mRNA that had been cloned into the Pst 1 site of pBR322 by the homopolymer extension technique. The rat PTH cDNA^^ was cloned by the Okayama and Berg method. The bovine mRNA was isolated from normal parathyroid glands, and the human mRNA was isolated from parathyroid adenomas. The sequence of the rat mRNA has been derived partially from the rat cDNA and from the sequence of the cloned gene. Kronenberg et al initially determined the sequence of a bovine cDNA clone, pPTHml, which contained about 60% of the PTH mRNA, including the entire region coding for pre-ProPTH. Restriction analysis of near full-length double-stranded cDNA, synthesized enzymatically from partially purified bovine PTH mRNA, indicated that about 200 nucleotides from the 3' untranslated region were missing in the clone. Analysis of several additional bovine PTH cDNA clones and the sequencing of cDNA of the 5' terminus of PTH mRNA, which was synthesized by extension of a primer with reverse transcriptase, provided the full bovine DNA sequence.^ Nucleotide sequences of the parathyroid (PTH) gene of 12 species of non-human primates belonging to suborder Anthropoidea were characterized.^^ The deduced amino acid sequences of exons II and III of the PTH gene of the 12 species of nonhuman primates was compared to the human PTH and revealed no amino acid substitution in the mature PTH among orangutans, chimpanzees, and humans. The results indicated that the PTH gene is highly conserved among primates, especially between great apes and humans.^^ The 5' end of the bovine mRNA sequence, which was determined by sequencing DNA complementary to the 5' end of PTH mRNA produced by primed reverse transcription, produced multiple 5' termini of the mRNA. The heterogeneity at the beginning of the 5' end of the mRNA was confirmed by SI nuclease mapping. The longest reverse transcribed cDNA was isolated and sequenced. Surprisingly, this cDNA contained a canonical TATA sequence at the beginning, which was in the proper position to direct the transcription of the shorter mRNAs. This result suggested that a second TATA sequence would be present 5' to the one detected in the cDNA and would direct the synthesis of the longer mRNAs. The predicted second TATA sequence was discovered when the gene was sequenced. The 5' end of the rat PTH mRNA was also analyzed by SI nuclease mapping and was less heterogeneous than the bovine mRNA. The single species of rat PTH mRNA corresponded to the larger of the bovine mRNAs. The size of the human mRNA, based on the cDNA sequence, is about 100 nucleotides longer than the bovine and rat mRNAs (Table 1). Northern blot analysis of the mRNAs was consistent with these predicted sizes. ̂ ^ The 3' untranslated region (UTR) of the avian PTH mRNA is 1236 nt long, much larger than any of the PTH mRNA 3'-UTRs (Table 1). In general the difference in size in 12 Molecular Biology of the Parathyroid Table 1, List of the known sequences for the PTH gene and the sizes of the mRNA, 5'-UTR, coding region and 3'-UTR NCBI Accession Number mRNA 5'UTR CDS 3'UTR Mus musculus Af066074: gene, exon 1 Af066075: gene, exons 2 and 3 and complete mRNA (deduced) 714 127 348 239