Mechanism of Endoplasmic Reticulum Retention of Mutant Vasopressin Precursor Caused by a Signal Peptide Truncation Associated with Diabetes Insipidus

Abstract

Autosomal dominant neurohypophyseal diabetes insipidus is caused by mutations in the gene encoding the vasopressin precursor protein, prepro-vasopressin-neurophysin II. We analyzed the molecular consequences of a mutation (ΔG227) recently identified in a Swiss kindred that destroys the translation initiation codon. In COS-7 cells transfected with the mutant cDNA, translation was found to initiate at an alternative ATG, producing a truncated signal sequence that was functional for targeting and translocation but was not cleaved by signal peptidase. The mutant precursor was completely retained within the endoplasmic reticulum. The uncleaved signal did not affect folding of the neurophysin portion of the precursor, as determined by its protease resistance. However, formation of disulfide-linked aggregates indicated that it interfered with the formation of the disulfide bond in vasopressin, most likely by blocking its insertion into the hormone binding site of neurophysin. Preventing disulfide formation in the vasopressin nonapeptide by mutation of cysteine 6 to serine was shown to be sufficient to cause aggregation and retention. These results indicate that the ΔG227 mutation induces translation of a truncated signal sequence that cannot be cleaved but prevents correct folding and oxidation of vasopressin, thereby causing precursor aggregation and retention in the endoplasmic reticulum. Autosomal dominant neurohypophyseal diabetes insipidus is caused by mutations in the gene encoding the vasopressin precursor protein, prepro-vasopressin-neurophysin II. We analyzed the molecular consequences of a mutation (ΔG227) recently identified in a Swiss kindred that destroys the translation initiation codon. In COS-7 cells transfected with the mutant cDNA, translation was found to initiate at an alternative ATG, producing a truncated signal sequence that was functional for targeting and translocation but was not cleaved by signal peptidase. The mutant precursor was completely retained within the endoplasmic reticulum. The uncleaved signal did not affect folding of the neurophysin portion of the precursor, as determined by its protease resistance. However, formation of disulfide-linked aggregates indicated that it interfered with the formation of the disulfide bond in vasopressin, most likely by blocking its insertion into the hormone binding site of neurophysin. Preventing disulfide formation in the vasopressin nonapeptide by mutation of cysteine 6 to serine was shown to be sufficient to cause aggregation and retention. These results indicate that the ΔG227 mutation induces translation of a truncated signal sequence that cannot be cleaved but prevents correct folding and oxidation of vasopressin, thereby causing precursor aggregation and retention in the endoplasmic reticulum. autosomal dominant neurohypophyseal diabetes insipidus neurophysin endoplasmic reticulum endo-β-N-acetylglucosaminidase H phosphate-buffered saline N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine Human neurohypophyseal diabetes insipidus is a disease characterized by a severe disturbance of antidiuresis becauseof the lack of the hypothalamic/neurohypophyseal antidiuretic hormone arginine-vasopressin. Patients produce great amounts of dilute urine and accordingly must drink large volumes of fluid to avoid exsiccosis. Neurohypophyseal diabetes insipidus most often results from trauma (including brain operations) or infiltrative or metastatic processes that damage the hypothalamus or the neurohypophysis, or it occurs as a primary idiopathic disorder. More rarely, the disease is hereditary and generally transmitted in an autosomal dominant manner (autosomal dominant neurohypophyseal diabetes insipidus, ADNDI) (1Baylis P.H. Robertson G.L. Postgrad. Med. J. 1981; 57: 36Crossref PubMed Scopus (34) Google Scholar,2Pedersen E.B. Lamm L.U. Albertsen K. Madsen M. Bruun-Petersen G. Henningsen K. Friedrich U. Magnusson K. Q. J. Med. 1985; 57: 883-896PubMed Google Scholar).1 Various mutations were identified in the coding sequence of the prepro-vasopressin-neurophysin II gene (3Rittig S. Robertson G.L. Siggaard C. Kovacs L. Gregersen N. Nyborg J. Pedersen E.B. Am. J. Hum. Genet. 1996; 58: 107-117PubMed Google Scholar). As illustrated in Fig.1 A, the precursor protein consists of four main segments (4Sausville E. Carney D. Battey J. J. Biol. Chem. 1985; 260: 10236-10241Abstract Full Text PDF PubMed Google Scholar): 1) a 19-amino acid signal sequence for targeting to the endoplasmic reticulum (ER); 2) the nonapeptide hormone vasopressin; 3) neurophysin II (NPII) consisting of 93 residues, which serves as the vasopressin transport protein in the circulation; and 4) a 39-amino acid glycopeptide of unknown function with a singleN-glycosylation site. The signal sequence is cleaved off by signal peptidase upon translocation of the precursor into the ER lumen. After folding and disulfide bond formation, pro-vasopressin-NPII passes through the Golgi apparatus into secretory granules where the other segments are separated by proteolytic removal of the linker residues. The mutations identified in different kindred of ADNDI are located in the sequences encoding the NPII moiety, the signal peptide, and in one case the vasopressin sequence (3Rittig S. Robertson G.L. Siggaard C. Kovacs L. Gregersen N. Nyborg J. Pedersen E.B. Am. J. Hum. Genet. 1996; 58: 107-117PubMed Google Scholar, 5Ueta Y. Taniguchi S. Yoshida A. Murakami I. Mitani Y. Hisatome I. Manabe I. Sato R. Tsuboi M. Ohtahara A. Nanba E. Shigemasa C. J. Clin. Endocrinol. Metab. 1996; 81: 1787-1790PubMed Google Scholar, 6Hansen L. Rittig S. Robertson G.L. Trends Endocrinol. Metab. 1997; 8: 363-372Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 7Gagliardi P.C. Bernasconi S. Repaske D.R. J. Clin. Endocrinol. Metab. 1997; 82: 3643-3646PubMed Google Scholar, 8Calvo B. Bilbao J.R. Urrutia I. Eizaguirre J. Gaztambide S. Castano L. J. Clin. Endocrinol. Metab. 1998; 83: 995-997Crossref PubMed Scopus (35) Google Scholar, 9Heppner C. Kotzka J. Bullmann C. Krone W. Muller-Wieland D. J. Clin. Endocrinol. Metab. 1998; 83: 693-696Crossref PubMed Scopus (37) Google Scholar). Typically, symptoms of ADNDI develop gradually over a period of months to years after birth (10Ellerman M. Acta Psychiatr. Neurol. 1939; 14: 233-241Crossref Scopus (3) Google Scholar, 11Levinger E.L. Escamilla R.F. J. Clin. Endocrinol. Metab. 1955; 15: 547Crossref PubMed Scopus (14) Google Scholar, 12Kaplowitz P.B. D'Ercole J. Robertson G.L. J. Pediatr. 1982; 100: 76-81Abstract Full Text PDF PubMed Scopus (38) Google Scholar, 13McLeod J.F. Kovacs L. Gaskill M.B. Rittig S. Bradley G.S. Robertson G.L. J. Clin. Endocrinol. Metab. 1993; 77: 599ACrossref PubMed Scopus (95) Google Scholar). Postmortem studies revealed degeneration of the hypothalamic magnocellular neurons in the nucleus supraventricularis and paraopticus, which synthesize the vasopressin-NPII precursor (14Hanhart E. Handbuch der Erbbiologie des Menschen. 4. Julius Springer, Berlin, Germany1940: 798-823Google Scholar, 15Gaupp R. Z. Neurol. Psychiatrie. 1941; 171: 514-546Crossref Scopus (9) Google Scholar, 16Forssmann H. Acta Med. Scand. Suppl. 1945; 159: 1-196Google Scholar, 17Braverman L.E. Mancini J.P. McGoldrick D.M. Ann. Intern. Med. 1965; 63: 503-508Crossref PubMed Scopus (97) Google Scholar, 18Green J.R. Buchan G.C. Alvord E.C. Swanson A.G. Brain. 1967; 90: 707-714Crossref PubMed Scopus (83) Google Scholar, 19Bergeron C. Kovacs K. Ezrin C. Mizzen C. Acta Neuropathol. 1991; 81: 345-348Crossref PubMed Scopus (86) Google Scholar). Based on these findings, it has been postulated that impaired transport and/or processing of the mutant precursor may result in its intracellular accumulation, eventually leading to degeneration of the vasopressinergic neurons and to the gradual manifestation of clinical symptoms. A recent study provided evidence for the toxicity of mutant precursors or their degradation products to cultured neuronal cells (20Ito M. Jameson J.L. Ito M. J. Clin. Invest. 1997; 99: 1897-1905Crossref PubMed Scopus (155) Google Scholar). We have investigated the molecular mechanism of an unusual mutation associated with ADNDI recently identified in a Swiss kindred (21Rutishauser J. Böni-Schnetzler M. Böni J. Wichmann W. Huisman T. Vallotton M.B. Froesch E.R. J. Clin. Endocrinol. Metab. 1996; 81: 192-198PubMed Google Scholar). Guanosine 227, the third nucleotide of the translation initiation codon of the vasopressin precursor gene, is deleted on one allele in affected family members. These heterozygous patients are completely vasopressin-deficient and show an abnormal appearance of the neurohypophysis in magnetic resonance imaging. Because loss of expression of one allele is not a plausible cause for the observed dominant phenotype, we hypothesized that a second, in-frame ATG present four codons downstream might serve as an alternative initiation site generating a mutant precursor with a truncated signal sequence (Fig.1 B, Vm). To test this hypothesis and to analyze how a signal sequence truncation might affect the fate of the protein, we transiently transfected mutant or wild-type prepro-vasopressin-NPII cDNA into COS-7 cells and characterized the effects of the guanosine 227 deletion on expression, membrane translocation, signal cleavage, polypeptide folding, and secretion of the precursor. The protein was found to be synthesized and translocated into the ER. However, the truncated signal sequence was not cleaved and interfered specifically with the folding and disulfide bond formation of the vasopressin nonapeptide portion of the precursor, thus causing the formation of disulfide aggregates and retention within the ER. The full-size cDNA of wild-type prepro-vasopressin-NPII was a gift from Dr. M. Ito (Northwestern University, Chicago, IL). To construct the full-size cDNA of the guanosine 227 deletion mutant, Vm, the 5′ end of the precursor cDNA up to the SmaI site was amplified by polymerase chain the site by the deletion of guanosine 227 is and a to a sequence in the The was at the SmaI site to the portion of the The cDNA was by In the a cDNA was identified with an deletion of codons of pro-vasopressin-NPII of chain of the precursor cDNA the in with a in the sequence was to the which the vasopressin precursor with a truncated signal sequence and with of the signal to 6 was to serine by polymerase chain of the 5′ portion of the wild-type precursor cDNA the in with a in the the portion was amplified the and a The the codon of cysteine 6 to the sequence thus four to a site M. N. 1998; Scholar). The polymerase chain products were at this site. the four were deleted by the with the with and producing the sequence encoding The were by COS-7 cells were in with at with The were with and of COS-7 cells and was to PubMed Scopus Google in cells were after in transfected cells were for in and for at with in cells were in cells and were to a and protein with endo-β-N-acetylglucosaminidase H were for in of were with of H for at were in and analyzed by were in and on were The of wild-type and mutant prepro-vasopressin-NPII were into the in with and with as J.P. M. Biol. 1991; PubMed Scopus Google Scholar). products were with for and with for After the were through a J.P. M. Biol. 1991; PubMed Scopus Google separated by and to The the protein was and to degradation an The at were analyzed by of signal peptidase was by in at a of and M. To membrane of wild-type and mutant vasopressin precursor, cells were and to as by and R. G. 1985; Full Text PDF PubMed Scopus Google Scholar). cells were with in at for J.P. M. Biol. 1991; PubMed Scopus Google Scholar). and was up in and to To analyze disulfide formation, transfected cells were with for at and were in and with with or and To the folding of wild-type and mutant vasopressin precursor, protein was with or and protein The were for at with The protease was by the of the was and the protein was analyzed by and cells were on with for at in and with in were with for binding was with The cells were with II primary in for with and with in for After with and the were in and analyzed a the cells were with II and a A. J. S. J. Biol. PubMed Scopus Google by with and of guanosine 227 in the prepro-vasopressin-NPII gene of the Swiss ADNDI kindred the translation initiation codon. of a mutant protein upon initiation at an alternative in-frame are ATG sequences in the prepro-vasopressin-NPII cDNA, encoding codon of and codon of the signal initiation at a a protein with most of the signal truncated by four The wild-type signal sequence is unusual in that it a and in its G. J. Biol. PubMed Scopus Google Scholar, T. A. J. Biol. PubMed Scopus Google Scholar). In the truncated signal the is To test translation of the mutant cDNA at one of the downstream and insertion into the ER wild-type and mutant were transiently in transfected COS-7 with for and with an the NPII portion of the precursor protein As the wild-type vasopressin precursor was synthesized as a of A, to with H it to be and thus translocated into the ER a of a was to H and the which was to that of the it to be an of the precursor The mutant cDNA with the guanosine 227 deletion was and The with an molecular of that was to by that in the of the codon a mutant protein with a functional signal sequence was to the wild-type protein, a was in Because is other ATG of the coding initiation must have at codon of the encoding the signal The molecular that the signal was not To the sequence of Vm, its cDNA was in and in the of for after The mutant precursor with was in the of A, As it the wild-type precursor 4) but with the as the wild-type precursor in the of a signal peptidase was synthesized with and the products were to was in and in the prepro-vasopressin-NPII this with the sequence with which has at and from the and of the precursor that translation at and that the truncated signal was not To analyze their secretion wild-type and mutant prepro-vasopressin-NPII precursors were in COS-7 for with for up to and from the and from the cells The wild-type precursor was into the with a of Because cells lack a secretory and the processing pro-vasopressin-NPII was The protein was to H and of of the from to in the Golgi In the mutant precursor was completely retained the cells in an of the mutant protein by with the of the ER through the for the ER protein A. J. S. J. PubMed Google and for mutant vasopressin precursor in the of cells the wild-type protein the of the precursor in the secretory the of the ER and a of the Golgi apparatus of retained mutant vasopressin precursor. COS-7 cells were and to as The wild-type precursor of the secretory ER and mutant prepro-vasopressin-NPII a of the ER. were for for mutant prepro-vasopressin-NPII and the ER signal cause ER retention by the protein in the ER was by of cells were at to and associated and As wild-type vasopressin precursor was in the and of the a membrane protein, was with the and The mutant vasopressin precursor was as as as the wild-type protein and that it was not in the of the ER. The uncleaved signal might cause ER retention by with correct folding and disulfide of the precursor. disulfide of disulfide is often in the of a protein To transfected cells were and at with to to precursor was and into that were in with or the wild-type precursor in its after of the disulfide and In the mutant precursor was at the in the but as aggregates that or did not the at aggregates are observed for T. A. J. Biol. PubMed Scopus Google Scholar). To the folding of the protein, wild-type and mutant precursors were to a protease protein was and protein and was with of The were and was analyzed by and the wild-type precursor a of 3) was that was completely to at up to to that of the NPII that the segments and were cleaved off at the by processing in secretory of and and 2) were at the that these to and vasopressin-NPII was by the an the vasopressin nonapeptide not the full-size precursor and the 2) were that the vasopressin of the mutant precursor with the NPII was as it was to the protease of 1) the uncleaved signal and vasopressin, and of the glycopeptide portion were The was the one to be the that it consists of the uncleaved vasopressin, and is the of the wild-type precursor that it an in the signal The uncleaved signal thus to affect the of the vasopressin to it did not with the folding of NPII In the precursor a of with an deletion of codons of pro-vasopressin-NPII of did not thus serves as a that protease on NPII In the wild-type are in disulfide in NPII and one in The mutant precursor, an in the uncleaved signal (Fig.1 To test this cysteine is for the formation of disulfide aggregates and we the mutant in which in the truncated signal sequence of was to As shown in mutation of this cysteine did not affect the of the was synthesized by cells as a protein A, and that the truncated signal cysteine was functional and The of the protein was retained in an within the After of was from the as protein, which is for of the NPII correct folding of NPII of did not most of the protein from disulfide-linked aggregates that aggregation is or not caused by The protease of the NPII portion in and that its disulfide were the disulfide bond in the vasopressin may not be in the mutant precursors and cause The of vasopressin, a disulfide with is that lack of signal in and with the formation of this disulfide its for oxidation to To test of vasopressin might cause ER we the mutant was to serine in the of the wild-type precursor its disulfide the signal is expression in an of the wild-type precursor A, of signal it was not from the cells and to H The mutation did not with folding of the NPII of was in the protease However, was found as aggregates analyzed These results indicate that to the disulfide bond in vasopressin is sufficient to cause aggregation and ER retention of the precursor The guanosine 227 deletion in thus the translation initiation it induces translation of a truncated signal sequence it cannot be prevents the correct folding and oxidation of is sufficient to cause aggregation and ER retention. In this we have analyzed the mechanism by which a mutation in the translation initiation codon of prepro-vasopressin-NPII ADNDI in a Swiss The mutant precursor was and that translation at an alternative ATG codon and a functional signal for ER targeting and translocation The initiation site is codon generating a signal sequence truncated by four residues. signal is in with the of ER targeting an unusual is The of the truncation was the loss of signal cleavage, which is from a molecular of is not by which mechanism signal peptidase was The to signal G. 14: PubMed Scopus Google Scholar, H. J. S. G. 1997; PubMed Scopus Google which are on of the result for the wild-type and the mutant The main are the to the site and which have and and the to the of the These are not affected by the However, it is that the mutant signal is within the translocation the with is As a of the site for signal peptidase might be I. G. J. Biol. PubMed Scopus Google evidence for a on signal on the of the signal of the on signal has been observed for II chain and the are II membrane with uncleaved signal of the in signal at that are not to signal peptidase J. B. Full Text PDF PubMed Scopus (83) Google Scholar, M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). in the of a signal sequence affect the of the signal in the insertion is not it is not in the signal is at the of the translocation or within the The uncleaved mutant vasopressin precursor was retained in the ER in a and and be in disulfide-linked aggregates of or T. A. J. Biol. PubMed Scopus Google Scholar). the in the precursor are in disulfide for one in the signal However, folding of the NPII which of the disulfide in the precursor, was not affected by the uncleaved the NPII was found to be as to as in the wild-type protein Based on the of the and the of NPII in the L. J.P. E. D. J. M. U. S. A. 1991; PubMed Scopus Google the glycopeptide is not in with the signal vasopressin segments and is thus to be In it not and thus cannot be for disulfide retention and aggregation is caused by the uncleaved signal and/or by of the signal sequence is not its mutation to serine did not aggregation and did not secretion results that the disulfide bond in vasopressin is not in the uncleaved mutant protein, the for disulfide with this we found that disulfide formation in vasopressin by one of its is sufficient to cause ER retention and the signal is The of NPII with a of vasopressin L. J.P. E. D. J. M. U. S. A. 1991; PubMed Scopus Google and of the neurophysin J.P. E. Biol. 1996; PubMed Scopus Google have been show that the of the hormone is in the binding and a and that the disulfide bond of the hormone is into the binding site. of the vasopressin of pro-vasopressin-NPII into the hormone binding site may be to the disulfide bond and by it from disulfide the signal sequence is not this is and the signal and vasopressin The that the NPII of the wild-type precursor is at that of the or and may of NPII the hormone is to its binding site. A of the on NPII has been in in folding studies R. E. 1996; PubMed Scopus Google Scholar). In the prepro-vasopressin-NPII precursor to different mutations causing ADNDI have been identified (3Rittig S. Robertson G.L. Siggaard C. Kovacs L. Gregersen N. Nyborg J. Pedersen E.B. Am. J. Hum. Genet. 1996; 58: 107-117PubMed Google Scholar, 5Ueta Y. Taniguchi S. Yoshida A. Murakami I. Mitani Y. Hisatome I. Manabe I. Sato R. Tsuboi M. Ohtahara A. Nanba E. Shigemasa C. J. Clin. Endocrinol. Metab. 1996; 81: 1787-1790PubMed Google Scholar, 6Hansen L. Rittig S. Robertson G.L. Trends Endocrinol. Metab. 1997; 8: 363-372Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 7Gagliardi P.C. Bernasconi S. Repaske D.R. J. Clin. Endocrinol. Metab. 1997; 82: 3643-3646PubMed Google Scholar, 8Calvo B. Bilbao J.R. Urrutia I. Eizaguirre J. Gaztambide S. Castano L. J. Clin. Endocrinol. Metab. 1998; 83: 995-997Crossref PubMed Scopus (35) Google Scholar, 9Heppner C. Kotzka J. Bullmann C. Krone W. Muller-Wieland D. J. Clin. Endocrinol. Metab. 1998; 83: 693-696Crossref PubMed Scopus (37) Google Scholar). the mutation to the signal was to in kindred (3Rittig S. Robertson G.L. Siggaard C. Kovacs L. Gregersen N. Nyborg J. Pedersen E.B. Am. J. Hum. Genet. 1996; 58: 107-117PubMed Google Scholar, 8Calvo B. Bilbao J.R. Urrutia I. Eizaguirre J. Gaztambide S. Castano L. J. Clin. Endocrinol. Metab. 1998; 83: 995-997Crossref PubMed Scopus (35) Google Scholar, 13McLeod J.F. Kovacs L. Gaskill M.B. Rittig S. Bradley G.S. Robertson G.L. J. Clin. Endocrinol. Metab. 1993; 77: 599ACrossref PubMed Scopus (95) Google Scholar, M. Y. T. K. H. T. M. J. Clin. Invest. 1993; PubMed Scopus Google Scholar, S. Kovacs L. Gregersen N. Robertson G.L. Pedersen E.B. D. Robertson G.L. the most ADNDI mutation In of by (3Rittig S. Robertson G.L. Siggaard C. Kovacs L. Gregersen N. Nyborg J. Pedersen E.B. Am. J. Hum. Genet. 1996; 58: 107-117PubMed Google C. Kotzka J. Bullmann C. Krone W. Muller-Wieland D. J. Clin. Endocrinol. Metab. 1998; 83: 693-696Crossref PubMed Scopus (37) Google and of by (3Rittig S. Robertson G.L. Siggaard C. Kovacs L. Gregersen N. Nyborg J. Pedersen E.B. Am. J. Hum. Genet. 1996; 58: 107-117PubMed Google have been These signal mutations the that are most for signal peptidase signal was in for the mutation M. Y. T. K. H. T. M. J. Clin. Invest. 1993; PubMed Scopus Google Scholar). These site are likely to cause ER retention by the mechanism as the guanosine 227 deletion mutant analyzed in this is one case of ADNDI with a mutation in vasopressin to L. Rittig S. Robertson G.L. Trends Endocrinol. Metab. 1997; 8: 363-372Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Because is for vasopressin binding to and of the mutant protein are likely to to the ER with the consequences as observed for the signal mutant analyzed other mutations were the NPII sequence and and mutations that most likely cause ER retention by the correct folding of the NPII The gradual of the dominant of ADNDI and studies that the disease is caused by the degeneration of the hypothalamic cells producing mutant hormone precursor. In a recent of different vasopressin precursor signal site mutant and NPII was observed (20Ito M. Jameson J.L. Ito M. J. Clin. Invest. 1997; 99: 1897-1905Crossref PubMed Scopus (155) Google that the retained precursor its degradation has a ER shown for the mutation of NPII G. D. H. Biol. 1996; 15: PubMed Scopus Google and the might be a of not prepro-vasopressin-NPII is of which in was found in in that were associated with damage S. C. J. Med. PubMed Scopus Google Scholar). mutant the were shown to be retained in the ER in a producing large of a PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In to this mutant vasopressin precursors were not found in but the ER and M. Jameson J.L. Ito M. J. Clin. Invest. 1997; 99: 1897-1905Crossref PubMed Scopus (155) Google Scholar). The disulfide aggregates to be that disulfide occurs to other in the ER by In the mutations that cause ADNDI are truncation of the third of NPII is likely to cause of the of the precursor, lack of signal in the guanosine 227 deletion mutant did not NPII folding the mechanism of by retained vasopressin precursors is the main in the molecular of ADNDI and may into the We C. A. and M. M. H. J. J. and M. B. Vallotton for and the of Dr. G. K. T. and Dr. for with in translation and protein A. for and L. and R. for the