Peptidomic Identification and Biological Validation of Neuroendocrine Regulatory Peptide-1 and -2

Abstract

Recent advances in peptidomics have enabled the identification of previously uncharacterized peptides. However, sequence information alone does not allow us to identify candidates for bioactive peptides. To increase an opportunity to discover bioactive peptides, we have focused on C-terminal amidation, a post-translational modification shared by many bioactive peptides. We analyzed peptides secreted from human medullary thyroid carcinoma TT cells that produce amidated peptides, and we identified two novel amidated peptides, designated neuroendocrine regulatory peptide (NERP)-1 and NERP-2. NERPs are derived from distinct regions of the neurosecretory protein that was originally identified as a product of a nerve growth factor-responsive gene in PC12 cells. Mass spectrometric analysis of the immunoprecipitate using specific antibodies as well as reversed phase-high performance liquid chromatography coupled with radioimmunoassay analysis of brain extract demonstrated the endogenous presence of NERP-1 and NERP-2 in the rat. NERPs are abundant in the paraventricular and supraoptic nuclei of the rat hypothalamus and colocalized frequently with vasopressin but rarely with oxytocin. NERPs dose-dependently suppressed vasopressin release induced by intracerebroventricular injection of hypertonic NaCl or angiotensin II in vivo. NERPs also suppressed basal and angiotensin II-induced vasopressin secretion from hypothalamic explants in vitro. Bioactivity of NERPs required C-terminal amidation. Anti-NERP IgGs canceled plasma vasopressin reduction in response to water loading, indicating that NERPs could be potent endogenous suppressors of vasopressin release. These findings suggest that NERPs are novel modulators in body fluid homeostasis. Recent advances in peptidomics have enabled the identification of previously uncharacterized peptides. However, sequence information alone does not allow us to identify candidates for bioactive peptides. To increase an opportunity to discover bioactive peptides, we have focused on C-terminal amidation, a post-translational modification shared by many bioactive peptides. We analyzed peptides secreted from human medullary thyroid carcinoma TT cells that produce amidated peptides, and we identified two novel amidated peptides, designated neuroendocrine regulatory peptide (NERP)-1 and NERP-2. NERPs are derived from distinct regions of the neurosecretory protein that was originally identified as a product of a nerve growth factor-responsive gene in PC12 cells. Mass spectrometric analysis of the immunoprecipitate using specific antibodies as well as reversed phase-high performance liquid chromatography coupled with radioimmunoassay analysis of brain extract demonstrated the endogenous presence of NERP-1 and NERP-2 in the rat. NERPs are abundant in the paraventricular and supraoptic nuclei of the rat hypothalamus and colocalized frequently with vasopressin but rarely with oxytocin. NERPs dose-dependently suppressed vasopressin release induced by intracerebroventricular injection of hypertonic NaCl or angiotensin II in vivo. NERPs also suppressed basal and angiotensin II-induced vasopressin secretion from hypothalamic explants in vitro. Bioactivity of NERPs required C-terminal amidation. Anti-NERP IgGs canceled plasma vasopressin reduction in response to water loading, indicating that NERPs could be potent endogenous suppressors of vasopressin release. These findings suggest that NERPs are novel modulators in body fluid homeostasis. Peptide hormones or neuropeptides function as cell-to-cell signaling molecules to mediate a variety of physiological phenomena. These bioactive peptides are cleaved from precursor proteins via limited cleavage and often undergo post-translational modifications to perform their functions (1Zhou A. Webb G. Zhu X. Steiner D.F. in with an of to the or a of peptides. have to the analysis of peptides in or body to the of a of previously uncharacterized peptides G. A. A. peptides identified are of in the to identify and hypothalamic peptides using specific the peptides identified have to be or or of abundant of peptide hormones or abundant molecules or the to identify peptides in candidates for novel bioactive peptides in in identification in peptidomics that we from the sequence of a to and the peptides for that to increase the of bioactive peptides to on peptides with a post-translational modification of bioactive peptides. we C-terminal amidation, shared by many bioactive peptides or peptide hormones peptides by a human of we two amidated peptides derived from the neurosecretory protein A. a precursor of bioactive peptides, are limited to C-terminal peptides as A. A. G. A. G. A. G. G. A. A. A. functions of peptides, identified by have not X. To a for their we peptides and to identify and cells in the rat. We of the well findings on the and of peptide hormones and the functions of peptides in to peptide a to the of bioactive of human medullary thyroid carcinoma TT cells in for was and using a as was to a Peptide to These to and by phase-high performance liquid regulatory phase-high performance liquid was analyzed by with a II and by of with a was to the and with and was also by G. peptides on an peptide using by and for by and of the peptides was on rat NERPs in the in and in and C-terminal to human and rat NERP-1 was with by the C-terminal of rat NERP-2 and human NERP-2 coupled with with with an of was as using and rat or of in was and rat or of the for NERP-1 and rat NERP-2 was with peptides with C-terminal C-terminal rat and human rat and human to a of A. G. A. G. A. G. G. A. A. A. and bioactive peptides amidated peptides. peptide angiotensin and of hypothalamus was and with a as previously of was by to was by and by to identify To endogenous of rat of rat brain and for NERP-1 and with antibodies and analyzed on a from TT extract was and analyzed as from in and on with to and and intracerebroventricular as for in of fluid and to and in with the for from the from with or for or and as previously from with with antibodies NERP-1 rat NERP-2 and vasopressin as A. with or that with of and vasopressin in the supraoptic and paraventricular from of water for by in with specific for to and of rat and vasopressin to of rat vasopressin as analyzed on an to that of water and an injection of peptide injection of hypertonic NaCl of of or of vasopressin was using an in the hypertonic or of and explants from the hypothalamus was as previously with modifications a of for as in by the of was a of to of NERPs on in of water for of NERP-1 or to plasma to of water body was to by injection a water loading, an injection of or and plasma vasopressin was are as of with analysis of and the to be of NERPs from of TT analyzed peptides secreted from human medullary thyroid carcinoma TT to the amidated peptide hormones peptide and of the in a for was and to chromatography to a to using and peptide was by to of was analyzed with spectrometric for We identified amidated peptides of peptides the or to peptide and identification of a of peptide peptides with with the that peptide we two novel amidated peptides with of and of derived from distinct regions of the neurosecretory protein from human and the from and of peptides also identified on their and physiological we designated peptides as neuroendocrine regulatory peptide (NERP)-1 and identification of of the peptides from of TT cells. NERP-1 and NERP-2 the by of amidated peptides. and identification of NERP-1 and NERP-2 by from the precursor and with by G. and and from the and and by cleavage of peptide in the the of or have the sequence of human and rat on the human and rat NERP-1 and rat in the as by and suggest that the rat precursor of amidated peptides as To rat endogenous peptides, we antibodies specific to the C-terminal of an to human and rat NERP-1 and of rat NERP-2 and human NERP-2 for We that the C-terminal but does not with peptide or the not rat C-terminal peptide or human C-terminal peptide indicating that not the and of NERP-1 and the of rat brain and as in the of not These and by analysis of using antibodies and on the of the we that rat peptides are derived from the precursor with the endogenous of rat NERP-1 and NERP-2 and rat hypothalamus to rat NERP-1 or NERP-2 on with TT extract also the that to human NERP-1 and NERP-2 These findings suggest that the and of NERPs as with amidated bioactive peptides secreted by cells endogenous of rat NERPs by and with an NERP-1 or NERP-2 from rat brain to the of NERP-1 and NERP-2 of rat a of from rat hypothalamus by coupled with and the of rat NERP-1 and abundant in the rat hypothalamus with of NERPs in the and the nuclei that produce vasopressin and G. G. that NERPs frequently colocalized with body fluid but rarely with the of NERPs was in and that of vasopressin was in the cells are to NERPs the of NERPs with vasopressin in and on we that we could a function of NERPs in the of vasopressin with vasopressin in the and of of the NERP-1 and NERP-2 in frequently with vasopressin but rarely with indicating the of NERPs with vasopressin in an of NERPs in the NERP-1 and NERP-2 in with vasopressin in the in the and water in and by the of vasopressin of These in and suggest that NERPs are in the of body fluid with and injection of hypertonic NaCl or plasma vasopressin in and was dose-dependently suppressed by injection of NERP-1 injection of the vasopressin with but was that of NERP-1 of NERP-2 was not NERP-1 NERP-2 suppressed vasopressin secretion increase in plasma vasopressin by water was also suppressed by NERP-1 or NERP-2 of or reversed plasma vasopressin induced by water that NERPs function as endogenous peptides to vasopressin we the in of NERPs on vasopressin secretion using hypothalamic NERP-1 suppressed basal and vasopressin secretion from the and NERP-2 was but or was not vasopressin secretion in in gene in the and of water analysis of A. of NERPs on rat plasma vasopressin in response to with hypertonic NaCl or intracerebroventricular of but to suppressed plasma vasopressin induced by water intracerebroventricular of or reversed plasma vasopressin induced by water to the identification of bioactive peptides, we analyzed peptides in the of peptides from the of that we an that amidated peptide hormones as or peptide a C-terminal a post-translational modification often shared by bioactive peptides we that bioactive peptides could be identified using to discover a of bioactive peptides as or to from the sequence of a peptide of NERPs in specific hypothalamic nuclei us to on their in to peptide and to with in and in originally identified in rat PC12 cells as a nerve growth factor-responsive a protein in A. in PC12 cells that the protein in and secreted the A. A. by a precursor for bioactive peptides A. antibodies the of the peptides have and to C-terminal peptides and in a on rat cells and and A. A. the to the identification of that by of and A. G. A. G. A. G. G. A. A. A. to peptidomics have the identification of peptides in brain or fluid X. peptides are to be a for A. G. D.F. for the peptides, peptides have demonstrated to be spectrometric for peptide in C-terminal as a modification that peptides could to abundant peptides or peptides, that amidated peptides from have to also be that NERP-2 have in of bioactive peptides that for human and rat NERP-2 are cleaved the and identification of also a of a and in in also be by endogenous peptides that peptidomics to in the and neurosecretory cells in the from cells in the from vasopressin secreted the to of NERPs frequently colocalized with vasopressin in the of the and with vasopressin in response to water that NERPs in the hypothalamic of plasma NERP-1 and NERP-2 suppressed vasopressin release by of hypertonic or in vivo. in NERPs also vasopressin release from the and with amidated IgGs canceled plasma vasopressin reduction in response to water loading, indicating that NERPs could be potent endogenous suppressors of vasopressin release by the of vasopressin are by and and to vasopressin are and by release of and and NaCl in vasopressin vasopressin secretion and or proteins of NERPs have not identified the of to and vasopressin release from the hypothalamus suggest that the or to vasopressin using of or to the of NERPs on to vasopressin the by NERPs vasopressin NERPs are novel bioactive peptides in body fluid to the and of the of focused peptidomics to the of bioactive peptides. of NERPs and their the for signaling as well as the physiological of NERPs in body fluid homeostasis. Peptide hormones or neuropeptides function as cell-to-cell signaling molecules to mediate a variety of physiological phenomena. These bioactive peptides are cleaved from precursor proteins via limited cleavage and often undergo post-translational modifications to perform their functions (1Zhou A. Webb G. Zhu X. Steiner D.F. in with an of to the or a of peptides. have to the analysis of peptides in or body to the of a of previously uncharacterized peptides G. A. A. peptides identified are of in the to identify and hypothalamic peptides using specific the peptides identified have to be or or of abundant of peptide hormones or abundant molecules or the to identify peptides in candidates for novel bioactive peptides in in identification in peptidomics that we from the sequence of a to and the peptides for We that to increase the of bioactive peptides to on peptides with a post-translational modification of bioactive peptides. we C-terminal amidation, shared by many bioactive peptides or peptide hormones peptides by a human of we two amidated peptides derived from the neurosecretory protein A. a precursor of bioactive peptides, are limited to C-terminal peptides as A. A. G. A. G. A. G. G. A. A. A. functions of peptides, identified by have not X. To a for their we peptides and to identify and cells in the rat. We of the well findings on the and of peptide hormones and the functions of peptides in to peptide a to the of bioactive peptides. of human medullary thyroid carcinoma TT cells in for was and using a as was to a Peptide to These to and by phase-high performance liquid regulatory phase-high performance liquid was analyzed by with a II and by of with a was to the and with and was also by G. peptides on an peptide using by and for by and of the peptides was on rat NERPs in the in and in and C-terminal to human and rat NERP-1 was with by the C-terminal of rat NERP-2 and human NERP-2 coupled with with with an of was as using and rat or of in was and rat or of the for NERP-1 and rat NERP-2 was with peptides with C-terminal C-terminal rat and human rat and human to a of A. G. A. G. A. G. G. A. A. A. and bioactive peptides amidated peptides. peptide angiotensin and of hypothalamus was and with a as previously of was by to was by and by to identify To endogenous of rat of rat brain and for NERP-1 and with antibodies and analyzed on a from TT extract was and analyzed as from in and on with to and and intracerebroventricular as for in of fluid and to and in with the for from the from with or for or and as previously from with with antibodies NERP-1 rat NERP-2 and vasopressin as A. with or that with of and vasopressin in the supraoptic and paraventricular from of water for by in with specific for to and of rat and vasopressin to of rat vasopressin as analyzed on an to that of water and an injection of peptide injection of hypertonic NaCl of of or of vasopressin was using an in the hypertonic or of and explants from the hypothalamus was as previously with modifications a of for as in by the of was a of to of NERPs on in of water for of NERP-1 or to plasma to of water body was to by injection a water loading, an injection of or and plasma vasopressin was are as of with analysis of and the to be Mass of human medullary thyroid carcinoma TT cells in for was and using a as was to a Peptide to These to and by phase-high performance liquid regulatory phase-high performance liquid was analyzed by with a II and by of with a was to the and with and was also by G. reversed phase-high performance liquid chromatography angiotensin II neuroendocrine regulatory peptide paraventricular radioimmunoassay supraoptic reversed phase-high performance liquid chromatography Peptide peptides on an peptide using by and for by and of the peptides was on rat NERPs in the in and in and C-terminal to human and rat NERP-1 was with by the C-terminal of rat NERP-2 and human NERP-2 coupled with with with an of was as using and rat or of in was and rat or of the for NERP-1 and rat NERP-2 was with peptides with C-terminal C-terminal rat and human rat and human to a of A. G. A. G. A. G. G. A. A. A. and bioactive peptides amidated peptides. peptide angiotensin and of hypothalamus was and with a as previously of was by to was by and by to identify To endogenous of rat of rat brain and for NERP-1 and with antibodies and analyzed on a from TT extract was and analyzed as from in and on with to and and intracerebroventricular as for in of fluid and to and in with the for from the from with or for or and as previously from with with antibodies NERP-1 rat NERP-2 and vasopressin as A. with or that with of and vasopressin in the supraoptic and paraventricular from of water for by in with specific for to and of rat and vasopressin to of rat vasopressin as analyzed on an to that of water and an injection of peptide injection of hypertonic NaCl of of or of vasopressin was using an in the hypertonic or of and explants from the hypothalamus was as previously with modifications a of for as in by the of was a of to of NERPs on in of water for of NERP-1 or to plasma to of water body was to by injection a water loading, an injection of or and plasma vasopressin was are as of with analysis of and the to be of NERPs from of TT analyzed peptides secreted from human medullary thyroid carcinoma TT to the amidated peptide hormones peptide and of the in a for was and to chromatography to a to using and peptide was by to of was analyzed with spectrometric for We identified amidated peptides of peptides the or to peptide and identification of a of peptide peptides with with the that peptide we two novel amidated peptides with of and of derived from distinct regions of the neurosecretory protein from human and the from and of peptides also identified on their and physiological we designated peptides as neuroendocrine regulatory peptide (NERP)-1 and NERP-1 and rat in the as by and suggest that the rat precursor of amidated peptides as To rat endogenous peptides, we antibodies specific to the C-terminal of an to human and rat NERP-1 and of rat NERP-2 and human NERP-2 for We that the C-terminal but does not with peptide or the not rat C-terminal peptide or human C-terminal peptide indicating that not the and of NERP-1 and the of rat brain and as in the of not These and by analysis of using antibodies and on the of the we that rat peptides are derived from the precursor with the endogenous of rat NERP-1 and NERP-2 and rat hypothalamus to rat NERP-1 or NERP-2 on with TT extract also the that to human NERP-1 and NERP-2 These findings suggest that the and of NERPs as with amidated bioactive peptides secreted by cells endogenous of rat NERPs by and with an NERP-1 or NERP-2 from rat brain to the of NERP-1 and NERP-2 of rat a of from rat hypothalamus by coupled with and the of rat NERP-1 and abundant in the rat hypothalamus with of NERPs in the and the nuclei that produce vasopressin and G. G. that NERPs frequently colocalized with body fluid but rarely with the of NERPs was in and that of vasopressin was in the cells are to NERPs the of NERPs with vasopressin in and on we that we could a function of NERPs in the of vasopressin with vasopressin in the and of of the NERP-1 and NERP-2 in frequently with vasopressin but rarely with indicating the of NERPs with vasopressin in an of NERPs in the NERP-1 and NERP-2 in with vasopressin in the in the and water in and by the of vasopressin of These in and suggest that NERPs are in the of body fluid with and injection of hypertonic NaCl or plasma vasopressin in and was dose-dependently suppressed by injection of NERP-1 injection of the vasopressin with but was that of NERP-1 of NERP-2 was not NERP-1 NERP-2 suppressed vasopressin secretion increase in plasma vasopressin by water was also suppressed by NERP-1 or NERP-2 of or reversed plasma vasopressin induced by water that NERPs function as endogenous peptides to vasopressin we the in of NERPs on vasopressin secretion using hypothalamic NERP-1 suppressed basal and vasopressin secretion from the and NERP-2 was but or was not vasopressin secretion in in gene in the and of water analysis of A. of NERPs on rat plasma vasopressin in response to with hypertonic NaCl or intracerebroventricular of but to suppressed plasma vasopressin induced by water intracerebroventricular of or reversed plasma vasopressin induced by water of NERPs from of TT analyzed peptides secreted from human medullary thyroid carcinoma TT to the amidated peptide hormones peptide and of the in a for was and to chromatography to a to using and peptide was by to of was analyzed with spectrometric for We identified amidated peptides of peptides the or to peptide and identification of a of peptide peptides with with the that peptide we two novel amidated peptides with of and of derived from distinct regions of the neurosecretory protein from human and the from and of peptides also identified on their and physiological we designated peptides as neuroendocrine regulatory peptide (NERP)-1 and NERP-2. NERP-1 and rat in the as by and suggest that the rat precursor of amidated peptides as To rat endogenous peptides, we antibodies specific to the C-terminal of an to human and rat NERP-1 and of rat NERP-2 and human NERP-2 for We that the C-terminal but does not with peptide or the not rat C-terminal peptide or human C-terminal peptide indicating that not the and of NERP-1 and the of rat brain and as in the of not These and by analysis of using antibodies and on the of the we that rat peptides are derived from the precursor with the endogenous of rat NERP-1 and NERP-2 and rat hypothalamus to rat NERP-1 or NERP-2 on with TT extract also the that to human NERP-1 and NERP-2 These findings suggest that the and of NERPs as with amidated bioactive peptides secreted by cells NERPs abundant in the rat hypothalamus with of NERPs in the and the nuclei that produce vasopressin and G. G. that NERPs frequently colocalized with body fluid but rarely with the of NERPs was in and that of vasopressin was in the cells are to NERPs the of NERPs with vasopressin in and on we that we could a function of NERPs in the of vasopressin in the and water in and by the of vasopressin of These in and suggest that NERPs are in the of body fluid with and injection of hypertonic NaCl or plasma vasopressin in and was dose-dependently suppressed by injection of NERP-1 injection of the vasopressin with but was that of NERP-1 of NERP-2 was not NERP-1 NERP-2 suppressed vasopressin secretion increase in plasma vasopressin by water was also suppressed by NERP-1 or NERP-2 of or reversed plasma vasopressin induced by water that NERPs function as endogenous peptides to vasopressin we the in of NERPs on vasopressin secretion using hypothalamic NERP-1 suppressed basal and vasopressin secretion from the and NERP-2 was but or was not to the identification of bioactive peptides, we analyzed peptides in the of peptides from the of that we an that amidated peptide hormones as or peptide a C-terminal a post-translational modification often shared by bioactive peptides we that bioactive peptides could be identified using to discover a of bioactive peptides as or to from the sequence of a peptide of NERPs in specific hypothalamic nuclei us to on their in to peptide and to with in and in originally identified in rat PC12 cells as a nerve growth factor-responsive a protein in A. in PC12 cells that the protein in and secreted the A. A. by a precursor for bioactive peptides A. antibodies the of the peptides have and to C-terminal peptides and in a on rat cells and and A. A. the to the identification of that by of and A. G. A. G. A. G. G. A. A. A. to peptidomics have the identification of peptides in brain or fluid X. peptides are to be a for A. G. D.F. for the peptides, peptides have demonstrated to be spectrometric for peptide in C-terminal as a modification that peptides could to abundant peptides or peptides, that amidated peptides from have to also be that NERP-2 have in of bioactive peptides that for human and rat NERP-2 are cleaved the and identification of also a of a and in in also be by endogenous peptides that peptidomics to in the and neurosecretory cells in the from cells in the from vasopressin secreted the to of NERPs frequently colocalized with vasopressin in the of the and with vasopressin in response to water that NERPs in the hypothalamic of plasma NERP-1 and NERP-2 suppressed vasopressin release by of hypertonic or in vivo. in NERPs also vasopressin release from the and with amidated IgGs canceled plasma vasopressin reduction in response to water loading, indicating that NERPs could be potent endogenous suppressors of vasopressin release by the of vasopressin are by and and to vasopressin are and by release of and and NaCl in vasopressin vasopressin secretion and or proteins of NERPs have not identified the of to and vasopressin release from the hypothalamus suggest that the or to vasopressin using of or to the of NERPs on to vasopressin the by NERPs vasopressin NERPs are novel bioactive peptides in body fluid to the and of the of focused peptidomics to the of bioactive peptides. of NERPs and their the for signaling as well as the physiological of NERPs in body fluid homeostasis. to the identification of bioactive peptides, we analyzed peptides in the of peptides from the of that we an that amidated peptide hormones as or peptide a C-terminal a post-translational modification often shared by bioactive peptides we that bioactive peptides could be identified using to discover a of bioactive peptides as or to from the sequence of a peptide of NERPs in specific hypothalamic nuclei us to on their in to peptide and to with in and in originally identified in rat PC12 cells as a nerve growth factor-responsive a protein in A. in PC12 cells that the protein in and secreted the A. A. by a precursor for bioactive peptides A. antibodies the of the peptides have and to C-terminal peptides and in a on rat cells and and A. A. the to the identification of that by of and A. G. A. G. A. G. G. A. A. A. to peptidomics have the identification of peptides in brain or fluid X. peptides are to be a for A. G. D.F. for the peptides, peptides have demonstrated to be spectrometric for peptide in C-terminal as a modification that peptides could to abundant peptides or peptides, that amidated peptides from have to also be that NERP-2 have in of bioactive peptides that for human and rat NERP-2 are cleaved the and identification of also a of a and in in also be by endogenous peptides that peptidomics to in the and neurosecretory cells in the from cells in the from vasopressin secreted the to of NERPs frequently colocalized with vasopressin in the of the and with vasopressin in response to water that NERPs in the hypothalamic of plasma NERP-1 and NERP-2 suppressed vasopressin release by of hypertonic or in vivo. in NERPs also vasopressin release from the and with amidated IgGs canceled plasma vasopressin reduction in response to water loading, indicating that NERPs could be potent endogenous suppressors of vasopressin release. release by the of vasopressin are by and and to vasopressin are and by release of and and NaCl in vasopressin vasopressin secretion and or proteins of NERPs have not identified the of to and vasopressin release from the hypothalamus suggest that the or to vasopressin using of or to the of NERPs on to vasopressin the by NERPs vasopressin release. NERPs are novel bioactive peptides in body fluid to the and of the of focused peptidomics to the of bioactive peptides. of NERPs and their the for signaling as well as the physiological of NERPs in body fluid homeostasis. We and for