Proparathyroid Hormone Is Preferentially Cleaved to Parathyroid Hormone by the Prohormone Convertase Furin

Parathyroid hormone (PTH), an 84-amino acid peptide, is the major regulator of blood calcium homeostasis. Its mRNA, in addition to encoding the mature peptide, also encodes a “pre” sequence of 25 amino acids and a basic “pro” hexapeptide. To assess which of the subtilisin-like prohormone convertases can process proPTH to PTH we coinfected cells with a vaccinia virus construct expressing human preproPTH and vaccinia virus constructs expressing furin, PC1 or PC2. BSC-40 cells, having a constitutive secretory pathway, and GH4C1 cells, having a regulated secretory pathway, were used. PTH biosynthetic products in cell extracts and media were purified by high performance liquid chromatography, identified by radioimmunoassay, and unambiguously defined as either proPTH or PTH by ion-spray mass spectrometry. In both cell types, furin was the most effective in processing proPTH to PTH. In all cases only PTH was released into the medium. In addition, partially purified furin and PC1 were tested for their ability to appropriately cleave a tridecapeptide spanning the prohormone cleavage site found in proPTH. Here too furin was much more effective at cleaving at the correct site. Northern blot analysis and in situ hybridization showed that furin and preproPTH mRNA are co-expressed in the parathyroid, whereas PC1, PC2, and PC5 are not and PACE4 is expressed only at very low levels. Taken together these studies strongly suggest that furin is the enzyme responsible for the physiological processing of proPTH to PTH.

[1]  A. Rich,et al.  Nucleotide sequence of cloned cDNAs encoding human preproparathyroid hormone. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[2]  S. Takahashi,et al.  Purification and characterization of furin, a Kex2-like processing endoprotease, produced in Chinese hamster ovary cells. , 1992, The Journal of biological chemistry.

[3]  A. Rich,et al.  Pre-proparathyroid hormone: a direct translation product of parathyroid messenger RNA. , 1974, Proceedings of the National Academy of Sciences of the United States of America.

[4]  N. Seidah,et al.  The neuroendocrine precursor 7B2 is a sulfated protein proteolytically processed by a ubiquitous furin-like convertase. , 1994, The Journal of biological chemistry.

[5]  K. Nakayama,et al.  Positional and additive effects of basic amino acids on processing of precursor proteins within the constitutive secretory pathway , 1993, FEBS letters.

[6]  T Watanabe,et al.  Identification and functional expression of a new member of the mammalian Kex2-like processing endoprotease family: its striking structural similarity to PACE4. , 1993, Journal of biochemistry.

[7]  J. Habener,et al.  Calcium-dependent intracellular degradation of parathyroid hormone: a possible mechanism for the regulation of hormone stores. , 1975, Endocrinology.

[8]  N. Seidah,et al.  Proprotein conversion is determined by a multiplicity of factors including convertase processing, substrate specificity, and intracellular environment. Cell type-specific processing of human prorenin by the convertase PC1. , 1992, The Journal of biological chemistry.

[9]  J. Henion,et al.  The determination of protein, oligonucleotide and peptide molecular weights by ion-spray mass spectrometry. , 1988, Rapid communications in mass spectrometry : RCM.

[10]  N. Seidah,et al.  Enzymic characterization of murine and human prohormone convertase-1 (mPC1 and hPC1) expressed in mammalian GH4C1 cells. , 1993, The Biochemical journal.

[11]  K. Nakayama,et al.  Identification of the fourth member of the mammalian endoprotease family homologous to the yeast Kex2 protease. Its testis-specific expression. , 1992, The Journal of biological chemistry.

[12]  A. Rich,et al.  Proparathyroid hormone: identification of a biosynthetic precursor to parathyroid hormone. , 1972, Proceedings of the National Academy of Sciences of the United States of America.

[13]  Richard I. Joh,et al.  Identification of a second human subtilisin-like protease gene in the fes/fps region of chromosome 15. , 1991, DNA and cell biology.

[14]  I. Lindberg.  The new eukaryotic precursor processing proteinases. , 1991, Molecular endocrinology.

[15]  L. L. Chu,et al.  Calcemic fraction-A: biosynthetic peptide precursor of parathyroid hormone. , 1972, Proceedings of the National Academy of Sciences of the United States of America.

[16]  D. Steiner,et al.  Identification of a human insulinoma cDNA encoding a novel mammalian protein structurally related to the yeast dibasic processing protease Kex2. , 1990, The Journal of biological chemistry.

[17]  P. Barr,et al.  Mammalian subtilisins: The long-sought dibasic processing endoproteases , 1991, Cell.

[18]  H. Davidson,et al.  Proteolytic processing of chromogranin A in purified insulin granules. Formation of a 20 kDa N-terminal fragment (betagranin) by the concerted action of a Ca2+-dependent endopeptidase and carboxypeptidase H (EC 3.4.17.10). , 1987, The Biochemical journal.

[19]  H. Bennett,et al.  Purification of the two major forms of rat pituitary corticotropin using only reversed-phase liquid chromatography. , 1981, Biochemistry.

[20]  T. Watanabe,et al.  Arg-X-Lys/Arg-Arg motif as a signal for precursor cleavage catalyzed by furin within the constitutive secretory pathway. , 1991, Journal of Biological Chemistry.

[21]  M Chrétien,et al.  cDNA sequence of two distinct pituitary proteins homologous to Kex2 and furin gene products: tissue-specific mRNAs encoding candidates for pro-hormone processing proteinases. , 1990, DNA and cell biology.

[22]  B. Gibbs,et al.  Purification of antimicrobial peptides from an extract of the skin of Xenopus laevis using heparin-affinity HPLC: characterization by ion-spray mass spectrometry. , 1994, Analytical biochemistry.

[23]  J. Dixon,et al.  Isolation of two complementary deoxyribonucleic acid clones from a rat insulinoma cell line based on similarities to Kex2 and furin sequences and the specific localization of each transcript to endocrine and neuroendocrine tissues in rats. , 1991, Endocrinology.

[24]  L. Orci,et al.  Parathyroid hormone biosynthesis. Correlation of conversion of biosynthetic precursors with intracellular protein migration as determined by electron microscope autoradiography , 1979, The Journal of cell biology.

[25]  N. Seidah,et al.  Cloning and primary sequence of a mouse candidate prohormone convertase PC1 homologous to PC2, Furin, and Kex2: distinct chromosomal localization and messenger RNA distribution in brain and pituitary compared to PC2. , 1991, Molecular endocrinology.

[26]  A. Rich,et al.  Secretion of human parathyroid hormone from rat pituitary cells infected with a recombinant retrovirus encoding preproparathyroid hormone. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[27]  D. Steiner,et al.  Identification of a cDNA encoding a second putative prohormone convertase related to PC2 in AtT20 cells and islets of Langerhans. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[28]  A. Brake,et al.  cDNA and gene structure for a human subtilisin-like protease with cleavage specificity for paired basic amino acid residues. , 1991, DNA and cell biology.

[29]  N. Seidah,et al.  Proprotein and prohormone convertases of the subtilisin family Recent developments and future perspectives , 1992, Trends in Endocrinology & Metabolism.

[30]  N. Seidah,et al.  Electron microscopic immunocytochemical evidence for the involvement of the convertases PC1 and PC2 in the processing of proinsulin in pancreatic beta-cells. , 1995, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[31]  D. Goltzman,et al.  Isolation and analysis of human parathyrin in parathyroid tissue and plasma. Use of reversed-phase liquid chromatography. , 1981, The Biochemical journal.

[32]  W. Sung,et al.  Recombinant human parathyroid hormone synthesized in Escherichia coli. Purification and characterization. , 1988, The Journal of biological chemistry.

[33]  H. Kronenberg,et al.  Importance of the propeptide sequence of human preproparathyroid hormone for signal sequence function. , 1988, The Journal of biological chemistry.

[34]  J. Habener,et al.  RAdioimmunoassay of human proparathyroid hormone: analysis of hormone content in tissue extracts and in plasma. , 1976, The Journal of clinical endocrinology and metabolism.

[35]  R. Jilka,et al.  Formation and secretion of fragments of parathormone. Identification of cleavage sites. , 1986, The Journal of biological chemistry.

[36]  W. Sung,et al.  Synthesis and characterization of extended and deleted recombinant analogues of parathyroid hormone-(1-84): correlation of peptide structure with function. , 1990, Biochemistry.

[37]  N. Seidah,et al.  PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[38]  M Chrétien,et al.  cDNA structure of the mouse and rat subtilisin/kexin-like PC5: a candidate proprotein convertase expressed in endocrine and nonendocrine cells. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[39]  H. Kronenberg,et al.  Gene encoding parathyroid hormone. Nucleotide sequence of the rat gene and deduced amino acid sequence of rat preproparathyroid hormone. , 1984, The Journal of biological chemistry.

[40]  G. Thomas,et al.  Intracellular trafficking and activation of the furin proprotein convertase: localization to the TGN and recycling from the cell surface. , 1994, The EMBO journal.

[41]  D. Steiner,et al.  The new enzymology of precursor processing endoproteases. , 1992, The Journal of biological chemistry.

[42]  M. Chrétien,et al.  Detection of endopeptidase activity and analysis of cleavage specificity using a radiometric solid-phase enzymatic assay. , 1991, Analytical biochemistry.