(Received for publication, September 27, 1994; and in revised form, November 7, 1994)
From the
A preproparathyroid hormone allele from a patient with familial isolated hypoparathyroidism was shown to have a single point mutation in the hydrophobic core of the signal sequence. This mutation, changing a cysteine to an arginine codon at the -8 position of the signal peptide, was associated with deleterious effects on the processing of preproparathyroid hormone to proparathyroid hormone in vitro. To examine the biochemical consequence(s) of this mutation, proteins produced by cell-free translation of wild-type and mutant cRNAs were used in assays that reconstitute the early steps of the secretory pathway. We find that the mutation impairs interaction of the nascent protein with signal recognition particle and the translocation machinery. Moreover, cleavage of the mutant signal sequence by solubilized signal peptidase is ineffective. The consequence of this mutation on processing and secretion of parathyroid hormone is confirmed in intact cells by pulse-chase experiments following transient expression of the mutant protein in COS-7 cells. The inability of the mutant signal sequence, however, to interfere with the targeting and processing of other secreted proteins does not support obstruction of the translocation apparatus as the mechanism underlying the dominant mode of inheritance of hypoparathyroidism in this family.
Proteins destined for residence within membranes or for
secretion contain hydrophobic amino-terminal sequences referred to as
signal sequences(1) . These sequences direct the nascent
polypeptides bound to ribosomes to form a functional junction with
rough endoplasmic reticulum (RER) ()membranes, thereby
assuring the translocation of the growing polypeptide chain into the
lumen of the endoplasmic reticulum and its subsequent cleavage by the
luminal-localized signal peptidase enzyme (for review, see (2) ).
The nascent secretory protein is localized to the endoplasmic reticulum via a targeting apparatus consisting of the signal recognition particle (SRP) and its membrane-bound receptor on RER. The SRP binds to the signal sequence as it emerges from the large ribosomal subunit. This results in a transient delay or even arrest of translation (3) aimed in preventing premature folding of the precursor protein. When the SRP-ribosome complex encounters the SRP receptor or ``docking protein,'' a series of reactions take place that result in the insertion of the nascent chain into the translocation site, release of SRP, resumption of translation, and initiation of translocation. GTP binding and its hydrolysis are required for these events to take place (4, 5, 6) . As the nascent polypeptide transverses the translocation channel (7, 8) and emerges into the lumen of the endoplasmic reticulum, it is modified further by the signal peptidase enzyme complex that catalyzes the endoproteolytic cleavage of the signal sequence.
Only a small number of natural mutations in human signal sequences have been reported to have direct correlation with defective secretion and associated pathological states(9, 10, 11) . We have described such a mutation in the signal peptide of one allele of preproparathyroid hormone (prepro-PTH) gene from a kindred with a form of familial isolated hypoparathyroidism(9) . This is an inherited metabolic disorder characterized by hypocalcemia and hyperphosphatemia resulting from lack of biologically active circulating PTH, the major calcium-regulating peptide. In this family, the disorder was inherited as an autosomal dominant trait(12) . The single point (T to C) mutation changed the codon at position -8 (signal peptide residues are numbered negatively starting from the site of cleavage toward the amino terminus) of the signal peptide of prepro-PTH from cysteine to arginine, thereby disrupting the hydrophobic core of the signal sequence (Fig. 1). Associated with this change was a dramatic impairment in the processing of the in vitro translated mutant prepro-PTH protein to pro-PTH by microsomal membranes(9) .
Figure 1:
Signal peptide
sequence of human prepro-PTH. Amino acids -25 to -1
constitute the signal peptide of wild-type and mutant (single and
double) forms of human prepro-PTH. The 6 residues following the signal
peptidase cleavage site (arrowhead) make up the pro sequence,
and the 84 amino acids of mature PTH follow. Residues -16 to
-5 (underlined) comprise the hydrophobic core of the
signal peptide. The described patient's mutation (Cys Arg
at the -8 position; single mutant) and the additional
substitution at the -10 position of the signal peptide (Ala
Arg; double mutant), are indicated by boldface. Numbersabove the amino acids indicate their position relative to
the signal peptidase cleavage site.
Which step(s) of the early secretory process is affected by this mutation is not readily evident. Conceivably it could preferentially affect one or all of the steps involved, such as binding to SRPs, targeting to the RER, translocation through the membrane, and proteolytic processing by signal peptidase. In this report, we have systematically examined each of these steps using mutant and wild-type forms of in vitro translated prepro-PTH proteins by assaying their interaction with components of these various processes. Moreover, the consequence of this mutation on processing and secretion of PTH was examined in intact cells by transient expression of the mutant protein in COS-7 cells. Finally, we have used a co-transfection assay to define the mechanism by which this specific mutation could cause clinical hypoparathyroidism in the presence of a second, apparently normal(9) , PTH allele.
Plasmids pWT83, pSM83, and pDM83, all containing prepro-PTH(1-83) sequences without the termination codon, were constructed by subcloning a HindIII-PstI fragment containing the prepro sequences from plasmids pWT84, pSM84, and pDM84, respectively, into plasmid SP-PTH(XbaI/Bal31/ClaI/#6) comprising mature PTH(1-83) sequences without the termination codon (15) .
In order to construct a plasmid encoding a protein of the same size as prepro-PTH but lacking a functional signal sequence, NcoI linkers were introduced into the SmaI site in the polylinker of SP-PTH(XbaI/Bal31/ClaI/#7) (15) , and a 225-base pair HaeIII-HindIII fragment derived from the same plasmid was ligated into the NcoI site. This introduced an ATG initiation codon followed by PTH sequences (51-82)-Ala in place of the signal sequence. This plasmid was then restricted with ClaI and HindIII and ligated to a DpnI-HindIII fragment isolated from plasmid SP-PTH(15) , containing sequences encoding PTH(1-84). The resulting plasmid (pRM84, for random mutant signal sequence), encoded Met-PTH(51-82)-Ala-Ser-PTH(1-84).
To introduce an N-linked glycosylation site into the PTH-coding sequence of plasmids pWT84 and pSM84, synthetic oligonucleotides, encoding Ser-Asn-Gly-Ser-Gly-Glu-Gly-Val-Glu-Ser, were ligated into the unique TaqI site of the prepro-PTH coding sequence (the underlined sequence indicates the consensus sequence for N-glycosylation). The resulting plasmids, pWT84(G) and pSM84(G), differed from pWT84 and pSM84, respectively, only by the insertion of 9 amino acids between residues Ser-17 and Met-18 of the mature PTH protein.
Figure 2:
Processing of normal and mutant
prepro-PTH. Autoradiogram of [H]leucine-labeled
proteins derived from the translation of cRNAs transcribed from
plasmids containing wild-type (pWT84), single mutant (pSM84), and
double mutant (pDM84) prepro-PTH cDNA. Translation was performed in the
rabbit reticulocyte lysate cell-free system in the absence (0 eq) or
presence of increasing amounts (4, 8, and 12 eq) of canine pancreatic
microsomal membranes.
Figure 3: Effect of SRP on translation. The cRNAs for wild-type (pWT84), single mutant (pSM84), double mutant (pDM84), random mutant (pRM84), and rabbit globin were translated in a wheat germ cell-free system in the absence (0) or presence of exogenous SRP (final concentration 0.02 (1) and 0.04 (2) A280 units/ml).
Figure 4: Translocation-competent binding to microsomal membranes. Truncated cRNAs missing the termination codon were transcribed from plasmids encoding wild-type, single mutant, and double mutant cDNAs and translated in rabbit reticulocyte lysate system. Translocation-competent binding to microsomal membranes was assessed by centrifugation of the membranes through either a physiological salt- or an EDTA-sucrose step cushion. Radiolabeled proteins in the pellets (A) and corresponding supernatants (B) are displayed.
Figure 5: Protease sensitivity of membrane-bound nascent chains. Wild type (pWT84) and single mutant (pSM84) cRNAs were translated in the rabbit reticulocyte lysate system in the presence (+) of dog pancreas microsomal membranes. After translation was completed, reactions were treated with proteinase K (20 µg/ml) with (+) or without(-) the addition of 1% Triton X-100. Radiolabeled translation products were immunoprecipitated and analyzed by SDS-PAGE.
Fig. 6shows that upon translation of pWT84(G)-transcribed cRNA in reactions supplemented with microsomal membranes, two translational products with PTH immunoreactivity were seen that were not present in the absence of membranes. The smaller product migrated with an apparent molecular weight slightly greater than that of authentic pro-PTH and was therefore felt to be pro-PTH(G). The second product, which migrated more slowly than prepro-PTH(G), was believed to be the glycosylated form of pro-PTH(G). This was confirmed by treatment with endoglycosidase H which, by removing carbohydrate on this peptide, shifted its position on an SDS-PAGE gel to that of pro-PTH(G). Interestingly, prepro-PTH(G) appeared to be processed much more efficiently by canine microsomal membranes than the unmodified form of the protein (compare Fig. 2and Fig. 6), and this may simply reflect the influence of the extended length or the specific structure of the nascent chain.
Figure 6: Glycosylation of wild-type and mutant prepro-PTH(G). Plasmids pWT84(G) and pSM84(G) were transcribed in vitro and translated in the rabbit reticulocyte lysate cell-free system in the absence (-M) or presence (+M) of canine microsomal membranes. Translation products were immunoprecipitated and treated with (+E) or without (-E) endoglycosidase H.
Translation of pSM84(G)-transcribed cRNA in the presence of membranes resulted in the appearance of three PTH-immunoreactive products, two of which co-migrated with pro-PTH(G) and its glycosylated form, while the third was consistent with the unprocessed prepro-PTH(G) form. Again, the addition of 9 amino acids to the mature PTH molecule resulted in a more efficient cleavage of the signal sequence by microsomal membranes as compared with the unmodified form (see Fig. 2and Fig. 6). Yet, the mutant signal sequence was once again processed less efficiently than the wild-type sequence, as indicated by the persistence of the unprocessed mutant prepro-PTH(G) form in the immunoprecipitated products. Once cleaved, however, pro-PTH(G) was glycosylated appropriately as confirmed by treatment of the reaction products with endoglycosidase H. The addition of carbohydrate to this moiety provides direct and independent evidence for its translocation, although significantly impaired, across the endoplasmic reticulum membranes. Furthermore, no larger glycosylated product was found that might have represented a protein that was translocated, glycosylated, and yet not cleaved by signal peptidase. Therefore, the uncleaved mutant prepro-PTH(G) was not delivered to the glycosylation machinery on the inner surface of the microsomal membranes.
Figure 7: Signal peptidase assay. Following translation of either pWT84- and pSM84- or pWT84(G)- and pSM84(G)-transcribed cRNAs in a wheat germ extract, signal peptidase assays were performed by mixing aliquots of translation mixture and signal peptidase prepared by directly solubilizing canine pancreatic rough microsomes.
Figure 8:
Expression of wild-type and mutant forms
of prepro-PTH in COS-7 cells. Plasmids pWT84, pSM84, and pDM84 were
transiently transfected into COS-7 cells. Five days following
transfection, the cells were pulse-labeled with
[S]methionine for 15 min. At the indicated times
following pulse-labeling, the media were removed, and cell extracts
were prepared. Both cell extracts (A) and media (B)
were immunoprecipitated using a PTH-specific antibody prior to SDS-PAGE
analysis. *, sample not processed.
WT52 was co-transfected into COS-7 cells with either wild-type or mutant prepro-PTH expression vector. Over-expression of the mutant precursor for up to 10 days did not interfere with the processing of prepro-PTH(1-52) (Fig. 9). Although these results may simply reflect lack of sensitivity of the system, they do not support global interference with protein processing at the microsomal membrane level as a consequence of overexpression of the mutant prepro-PTH form.
Figure 9:
Co-transfection of wild-type and mutant
prepro-PTH. Plasmids pWT84 and pSM84 were transfected into COS-7 cells
either alone or in combination with pWT52. After the indicated number
of days posttransfection, cells were pulse-labeled with
[S]methionine for 15 min. Cell extracts were
then prepared and immunoprecipitated prior to SDS-PAGE
analysis.
Translocation from the cytoplasm into the endoplasmic reticulum, is a multistep process requiring a functional amino-terminal signal peptide. A signal sequence must perform effectively several distinct functions required for the efficient translocation of secreted proteins. These subfunctions include its recognition and binding to SRP, its interaction with membrane-bound components of the export machinery, opening the protein-conducting channels to initiate translocation, and appropriate presentation to the signal peptidase for cleavage.
Three domains have been identified as a common feature of
eukaryotic signal sequences, and considered to be necessary for
carrying out these functions: a positively charged NH terminus, a central hydrophobic core of 10-15 amino acid
residues, and a polar COOH-terminal region(24, 25) .
While the COOH-terminal region influences the efficiency and fidelity
of signal peptidase cleavage, intactness of the hydrophobic region is
indispensable for initiating translocation.
Two reported inherited
mutations in the signal sequence of human secreted proteins, namely
preprovasopressin(10) , and preprofactor X (11) ,
involve the COOH-terminal region of the respective signal peptides.
Thus, a point mutation resulting in substitution of Arg for Gly at the
-3 position of the factor X signal peptide (Factor X ) blocks cleavage by signal peptidase but does not
interfere with targeting and translocation to the RER(11) .
Similarly, a naturally occurring substitution of Thr for Ala at the
-1 position of the signal peptide of preprovasopressin results in
central diabetes insipidus(10) . This mutant protein, similar
to the Factor X
, undergoes inefficient cleavage
by signal peptidase, although targeting and translocation to the RER
are not measurably affected.
The present study is the first to examine the effect of a naturally occurring substitution at the hydrophobic core of a signal peptide that results in human disease. Prepro-PTH, the precursor of PTH, contains a typical 25-residue amino-terminal signal sequence followed by a 6-residue prospecific peptide and the mature hormone (residues 1-84; see Fig. 1). The hydrophobic core of the human prepro-PTH signal peptide is composed of 12 contiguous uncharged amino acids (residues -5 to -16 of the signal peptide).
In the present study, we have demonstrated that substitution of a charged amino acid, Arg for Cys, in the signal peptide hydrophobic core of prepro-PTH impairs co-translational translocation as well as posttranslational cleavage by isolated signal peptidase. The impairment was even more evident when two charged residues were introduced in the hydrophobic core of the signal sequence. In contrast to deletion mutants(26) , this change interferes not only with translocation and cleavage by signal peptidase but also with binding to SRP. Since substitution of a hydrophobic amino acid, leucine, for cysteine or its deletion was ineffective in modifying translocation and processing (26) , the present findings would suggest that a change in the hydrophobicity of the core is responsible for the observed global disruption in the processing of the mutant prepro-PTH. Similarly, single charged amino acids introduced in the hydrophobic core of the E. coli maltose binding protein signal peptide impair secretion of the protein into the external periplasmic compartment of the cell(27) .
It is rather intriguing that three distinct reports of inherited mutations in the signal sequence of human secreted proteins, namely prepro-PTH(9) , preprovasopressin(10) , and preprofactor X(11) , demonstrate inheritance of the associated disorder in an autosomal dominant fashion. As is the case for the other two reported disorders, however, it remains unclear why individuals with a mutated PTH allele have hypoparathyroidism. From transfection studies in COS-7 cells, it would appear that expression of the mutant allele does lead to secretion of PTH, albeit inefficiently. Moreover, the normal allele would be expected to produce sufficient circulating PTH to maintain calcium homeostasis. The only other reported case of familial isolated hypoparathyroidism segregating with a mutation in the PTH gene, involved a point mutation affecting intron splicing and was associated with autosomal recessive inheritance of the disorder(28) . Since heterozygous individuals for this mutant allele were unaffected, it would appear that one normal PTH allele is sufficient for maintaining calcium homeostasis.
Hypopathyroidism in the presence of
one normal PTH allele would therefore suggest that the mutant gene
product exerts a dominant negative effect in vivo. The mutant
protein might interfere with the normal targeting and processing of
other secreted proteins, including the normal PTH precursor. Such
interference might even lead to destruction of parathyroid tissue in
affected individuals; unfortunately, this is difficult to evaluate
because the tissue is not readily accessible. Export incompatibility,
however, has been observed in E. coli expressing
transport-defective -galactosidase leading to lethal jamming of
the cellular export machinery(29) .
The phenotype of the single-mutant prepro-PTH suggests that it might have dominant negative effects under appropriate conditions. The mutation allows a fraction of the prepro-PTH precursor to enter the translocation machinery, but the mutant protein is then cleaved inefficiently by signal peptidase. The inability of the uncleaved protein to reach the glycosylation machinery (assessed using the precursor modified by inclusion of a glycosylation signal) suggests that the precursor is not transported fully across the microsomal membrane. Such a protein that engages the translocation apparatus but fails to move through the apparatus efficiently might well have dominant negative effects. The competition experiment in Fig. 9failed to demonstrate such an effect; perhaps higher levels of protein expression or a longer term experiment are needed. Nevertheless, the observation that all three reported human signal sequence mutations involve proteins that partly engage the secretory apparatus and appear to have dominant effects suggests that in vivo these mutant proteins cause dominant secretory dysfunction.