Procalcitonin Is a Glycoprotein*

Messenger RNA extracted from rat medullary carcinoma of the thyroid directs the synthesis in cell-free translation systems of a precursor of calcitonin, M, = 15,000, substantially larger than. the mature form of the hormone, M, = 3,500. When translations of the mRNA were carried out in the presence of microsomal mem- branes prepared from a canine pancreas, a larger product (apparent M, = 17,000) was observed by electropho- resis of the labeled proteins in the translation mixtures on sodium dodecyl sulfate-polyacrylamide gels. This membrane-processed product of M, = 17,000 was specifically immunoprecipitated by an antiserum to syn- thetic calcitonin and bound to concanavalin A-Sepha-rose. Incubation of the proteins synthesized in the cell- free translations performed in the presence of microsomal membranes with the glycosidase, endo-P-N-ace- tylglueosaminidase H, reduced the apparent molecular weight of the membrane-processed precursor from 17,000 to 12,000. In addition, the processed M, = 17,000 calcitonin-related precursor, but not the initial, un- processed precursor of M, = 15,000, was resistant to proteolytic digestion by a mixture of trypsin and chymotrypsin. These results indicate that the biosynthesis Immunoprecipitations-Immunoprecipitations were performed utilizing a double antibody immunoprecipitation procedure. A antiserum to human calcitonin established previously to cross-react potently with rat calcitonin’ was used in the experiments. Ten micro-liters of the translation was dissolved in 0.1 ml of a solution consisting of 10 mM NaH2P0.,, pH 7.6, 1 Na2EDTA, and 1% Triton X-100, and incubated overnight at 4°C with 1.0 pl of the antiserum to calcitonin. Then a goat antiserum against rabbit-y-globulin was added. After an 18-h incubation (4OC) with anti-rabbit goat antiserum, the immunoprecipitates were centrifuged at 10,000 rpm min and washed three times with phosphate-buffered saline, 1% (v/v) Triton The immunoprecipitates were dissolved sample and boiled min before electrophoresis on polyacrylamide-SDS gels.

sequence to the mRNA coding for the precursor (6). The structural analysis indicates that the sequence of calcitonin is located within a large precursor flanked at its amino and carboxyl ends by both basic amino acid residues, typical of prohormone cleavage sites, and by extensions consisting of cryptic peptide sequences. Presumably the co-and post-translational processing of calcitonin involves multiple proteolytic cleavages of the precursor, similar to the processing of other prehormones, including preproparathyroid hormone (7), preproinsulin (8), and the common precursor for ACTH, MSH, and the endorphins (9).
Evidence obtained from studies with cell-free systems indicates that certain modifications of protein precursors occur co-translationally on nascent chains. These include the glycosylation of secretory and membrane proteins (10)(11)(12), as well as the cleavage from the precursors of NH2-terminal leader, or signal, sequences (13)(14)(15). The functions of leader sequences appear to be in the transport of nascent secretory proteins into the cisternae of the rough endoplasmic reticulum. Cleavages of leader sequences from and glycosylation of, the nascent proteins occur when the chains are approximately 70 amino acids long (11,16,17). The biological functions of the carbohydrates attached to the nascent chains are unknown, particularly under circumstances in which certain sugars are cleaved from the carbohydrate complex during further cellular processing and transport of the proteins, as was noted for glycoproteins synthesized in Sindbis virus and vesicular stomatitus virus infected chicken embryo fibroblasts (18).
Preliminary evidence reported previously suggests that large immunoreactive forms of calcitonin, extracted from human medullary thyroid carcinomas, contain carbohydrate (19). Now we report that a membrane-dependent glycosylation of a biosynthetic precursor of calcitonin takes place during cell-free translations of mRNA carried out in the presence of microsomal membranes. In addition, we show that the endoglycosidase, endo-/3-N-acetylglucosaminidase H, provides structural information on the nature of the carbohydrate moiety attached to the calcitonin precursor.

EXPERIMENTAL PROCEDURES
Preparation of RNA-Rats of the WAG/Rij strain carrying a calcitonin-secreting medullary carcinoma of the thyroid transplanted beneath the renal capsule were obtained from the Institute for Experimental Gerontology, Rijswik, The Netherlands (20)(21)(22). Small sections of the tumor weighing less than 50 mg were transplanted beneath the left kidney capsule in normal 4-week-old rats of the same strain with the animals under light ether anesthesia. Six months (or more) later, tumors and omental metastases were removed from the rats under light ether anesthesia. The tumor tissue was placed immediately in liquid nitrogen and stored in this manner until used. Polyadenylated RNA was isolated from rat tissue using the procedures described by Kronenberg et al. (23) and Majzoub et al. (24). Approximately 50 pg (1.0 A260 unit) of polyadenylated RNA was obtained from 1 g of the tumor.

Procalcitonin Is
Cell-free Translations-A heterologous cell-free translation system was prepared from wheat germ (25). RNA (0.1 to 1.0 pg) dissolved in sterile H 2 0 was translated in reaction mixtures (20 to 100 pl) containing 1 mCi/ml of ~.-[''S]methionine (800-1,000 Ci/mmol, New England Nuclear, Boston, MA). Translation reactions were terminated by freezing or adding, to the reaction vessels, buffer used for electrophoresis consisting of 0.05 M Tris-HCI, pH 7.0, 1% sodium dodecyl sulfate (SDS). 1% /jl-mercaptoethanol, 10% glycerol, and 0. 19 bromphenol blue (electrophoresis sample buffer). In experiments involving membrane processing of translation products, 1.0 pl (115 units/ml) of microsomal membranes, prepared by the method of Katz et al. (26) from a dog pancreas, was added directly to the translation mixtures before the addition of the RNA.
Polyacclamide Gel Electrophoresis in Sodium Dodecyl Sulfute-Translation products, dissolved in electrophoresis sample buffer, were heated at 95°C for 2 min and applied to gradient polyacrylamide (10-20%) slab gels containing 0.lV SDS (27). Electrophoresis was performed a t a constant 100 V until the dye reached the bottom of the gel. Gel slabs were stained in 0.2%. Coomassie brilliant blue (Sigma), destained in 25% methanol, 7% acetic acid, dried in uucuo, and treated with a fluorography enhancer (Enhance, New England Nuclear). Autofluorograms were prepared by exposing the dried gels to Kodak SB-5 film for periods of 1 to 14 days. Unlabeled proteins of known molecular weights including bovine parathyroid secretory protein, M , = 70,000, actin, M , = 46,000, preproparathyroid hormone, M , = 14.000, bovine heart cytochrome c, M , = 12,400, proparathyroid hormone, M , = 10,300, and parathyroid hormone, M , = 9,500, were used as molecular weight markers.
Immunoprecipitations-Immunoprecipitations were performed utilizing a double antibody immunoprecipitation procedure. A rabbit antiserum to human calcitonin established previously to cross-react potently with rat calcitonin' was used in the experiments. Ten microliters of the translation mixture was dissolved in 0.1 ml of a solution consisting of 10 mM NaH2P0.,, pH 7.6, 1 mM Na2EDTA, and 1% Triton X-100, and incubated overnight a t 4°C with 1.0 pl of the antiserum to calcitonin. Then a goat antiserum against rabbit-yglobulin was added. After an 18-h incubation (4OC) with anti-rabbit goat antiserum, the immunoprecipitates were centrifuged a t 10,000 rpm for 15 min and washed three times with phosphate-buffered saline, 1% (v/v) Triton X-100. The immunoprecipitates were dissolved in sample electrophoresis buffer, heated to 37°C for 1 h, and boiled for 2 min before electrophoresis on polyacrylamide-SDS gels.
Limited Proteolysis Experiments-Aliquots of reaction mixtures from cell-free translations carried out with or without the addition of microsomal membranes were incubated for 60 min a t 0°C with a mixture of trypsin and chymotrypsin at a final concentration of 50 pg/ml of each of the enzymes. T o one-half of each reaction mixture, Triton X-100 was added to a final concentration of 1% before addition of the proteases. Proteolytic digestions were terminated by addition of Trasylol (FBA Pharmaceuticals) to a final concentration of 1,000 units/ml. Digestion urith Endoglycosiduse H-Aliquots of reaction mixtures from cell-free translations were adjusted to 100 mM sodium citrate, pH 6.0, 0.1 M 2-mercaptoethanol, 0.8% SDS, and heated for 2 min a t 100°C. After cooling, approximately 3 pg of endo-P-N-acetylglucosaminidase H (a generous gift of Dr. H. Green, Massachusetts Institute of Technology) was added, and the mixture was incubated for I6 h a t 37°C. The reaction was terminated by the addition of electrophoresis sample buffer and boiled a t l00'C for 2 min.
Concanavalin A Affinity Binding-Concanavalin A (Con A) coupled to agarose (Pharmacia) was extensively washed by alternating centrifugation (5 min, 12,000 rpm) and resuspension in buffer containing 1 mM MgC12, 1 mM MnC12, 1 mM CaC12, 200 mM NaCI, and 50 mM Tris, pH 7.4 (Con A buffer). To the washed Con A-Sepharose (approximately 100 pl of packed volume in a 0.5 ml of microfuge tube) was added 25 p1 of a cell-free translation mixture containing 1%. Triton X-100. After gentle mixing for 30 min at 4OC the Con A-Sepharose was washed with Con A buffer containing 1% Triton X-100. The material bound to the lectin was removed by either elution with Con A buffer containing 1% Triton and 0.2 M a-methylmannoside (12) or by boiling the Con A-Sepharose in electrophoresis sample buffer.

RESULTS
Polyadenylated RNA prepared from the rat medullary carcinoma of the thyroid directs the synthesis of two major a Glycoprotein polypeptides ( M , = 15,000 and 13,000) in wheat germ cell-free translation assays (Fig. 1, lane 4 ) . The M , = 15,000 product was shown previously to be a biosynthetic precursor of calcitonin (4). As shown in Fig. 1, the M , = 15,000 product is specifically immunoprecipitated by an antiserum against synthetic calcitonin (lane 3), and the immunoprecipitation is inhibited by the addition to the immunoprecipitation reaction of 10 pg of synthetic human calcitonin (lane 2).
To characterize further the cell-free product, the translations were performed in the presence of microsomal membranes. These membranes process secretory proteins by way of cleavages of leader, or signal sequences from the NHs terminus of these proteins, and, in some instances, by glycosylation of the nascent polypeptides (10)(11)(12)(13)(14)(15)(16)(17). Addition of microsomal membranes to the translation assay significantly altered the mobility of several of the RNA-directed translation products (Fig. 1, lane 5). The protein of M , = 15,000, as visualized by autofluorography of the SDS-polyacrylamide gels, is greatly diminished concomitant with the appearance of several new polypeptides (Mr = 17,000, 12,000, and 10,000).
When antiserum to synthetic human calcitonin was added to the membrane-reacted translation products, the M , = 17,000 protein is specifically immunoprecipitated (lanes 6,9, and IO). Two minor products of M , = 15,000 and 12,000 also appear in the immunoprecipitated products (Fig. 1, lane 6) and most probably represent small amounts of the unprocessed calcitonin precursor ( M , = 15,000) and of the calcitonin precursor from which a leader peptide of M, = 3,000 has been cleaved but has escaped glycosylation ( M , = 12,000).
To test the possibility that the M , = 17,000 represents a glycosylated form of procalcitonin, membrane-reacted translation products were incubated with concanavalin A-Sepharose, a lectin which specifically binds glycoproteins ( T o further test the possibility that the M , = 17,000 calcitonin-related translation product is a glycoprotein, we treated the translation products with the endoglycosidase, endo-P-Nacetylglucosaminidase H (endoglycosidase H). This enzyme cleaves from glycoproteins the di-N-acetylchitobiose sugar moiety linked to asparagine residues. In addition, for full activity, this enzyme requires the presence of at least 4 mannose residues linked to the di-N-acetylchitobiose (29). Treatment of the translation products synthesized in presence of membranes with endoglycosidase H resulted in the disappearance of the M , = 17,000 protein (Fig. 2, lane 4 ) and a corresponding enhancement in the intensity of labeling of a protein of M , = 12,000. This protein corresponds to the predicted migration of the nonglycosylated calcitonin precursor from which a leader sequence of M , = 3,000 was removed. Treatment, with endoglycosidase H, of the translation reactions performed in the absence of microsomal membranes had no effect on the mobility of any of the labeled products (Fig.  2, lanes 1 and 2 ) . Immunoprecipitation analyses using an antiserum against synthetic human calcitonin show that the protein of M , = 17,000, are related to calcitonin (Fig. 2, lanes  6-9). The presence of a small amount of the M , = 15,000, unprocessed calcitonin precursor in lanes 6 and 7 suggests that in this particular experiment, cleavage and/or glycosylation of the precursor was incomplete.
Evidence presently available indicates that glycosylation and cleavage of NH2-terminal leader sequences from nascent pol-ypeptides occur co-translationally during growth of the nascent chains (10-12). The glycoproteins are sequestered within microsomal vesicles (14-16). Such sequestration of glycoproteins can he tested for experimentally by digestion of the products of the cell-free translation with proteolytic enzymes. Sequestered proteins are resistant to digestion by mixtures of trypsin and chymotr-ypsin, whereas products that lie outside the microsomal vesicles are hydrolyzed by the proteases. As shown in Fig. 3

DISCUSSION
In these studies, we show that polyadenylated RNA prepared from a rat medullary carcinoma of the thyroid directs the synthesis in cell-free translations supplemented with microsomal membranes of a glycosylated precursor of calcitonin of apparent M , = 17,000. The M , = 17,000 tanslation product was shown to he structurally related to calcitonin by immunoprecipitation of the precursor with antisera directed against synthetic calcitonin. Inasmuch as the antiserum used in these studies was raised against synthetic calcitonin, it is highly unlikely that antibodies present in the antiserum recognized proteins that are not related to calcitonin. In addition the immunoprecipitation of the 17,000 M , protein was specifically inhibited by the presence of unlabeled, homogeneous synthetic calcitonin. Recently, we have obtained additional information about the structure of the calcitonin precursor by analysis of the nucleotide sequence of a cloned cDNA (6). The cDNA was shown to contain a nucleotide sequence coding for the complete amino acid sequence of calcitonin and to specifically hybridize with the mRNA coding for the 15,000 M,, and the processed 17,000 M,, proteins shown in Figs. 1-3. These studies confirm that the cell-free translation products immunoprecipitated by antisera to calcitonin are precursors of calcitonin.
Two distinct methods were used to determine that the M , = 17,000 calcitonin precursor contained carbohydrate; adsorption to concanavalin A-Sepharose and sensitivity of the oligosaccharides of the precursor to cleavage by the endoglyco- with the cleavage of carbohydrate from the precursor. In addition, -the glycosylated calcitonin precursor as judged by i t s resistance to proteolytic digestion was shown to he sequestered within microsomal vesicles after its synthesis, a finding consistent with the membrane-dependent processing and translocation of this glycoprotein into the vesicles. Studies from a number of laboratories indicate that the glycosylation of proteins occurs through the transfer of carbohydrate to nascent polypeptide chains via a dolichol-oligosaccharide intermediate localized in microsomal membranes (30-34). The carbohydrate transferred is a di-N-acetylchitibiose-mannose unit that can he subsequently extended by the terminal addition of other sugar residues (35,36). In the present studies, the nature of carbohydrate moiety attached to the calcitonin precursor was probed with a well character-ized enzyme, endo-P-N-acetylglucosaminidase H, that requires for its enzymatic activity the presence of a di-N-acetylchitobiose unit containing at least 4 mannose residues (29). In addition, the attachment of the dolichol sugar unit to the peptide chain requires an amino acid sequence of the form Asn-X-Ser (or Thr), with asparagine serving as the carbohydrate acceptor (37,38). From the above studies, a minimum structure of the carbohydrate moiety attached to the calcitonin precursor can be deduced, consisting of: mannose (greater than 3 residues) + N-acetylglucosamine --* N-acetylglucosamine + asparagine.
Although the complete structure of the calcitonin precursor is not yet available, the sequence of 80 of the approximately 130 amino acids has been deduced from the nucleotide sequence of a cloned cDNA (6). Based on this sequence, the only potential acceptor site for N-linked glycosylation of the form -Asn-X-Ser (Thr)-is found in the sequence of calcitonin itself; Asn-Leu-Ser at positions 3-5. This tripeptide sequence, which is strictly conserved in all calcitonin sequences (2, 39), lies in the center of an invariant "ring" structure formed by a disulfide bridge linking residues 1 and 7. The possible glycosylation of this distinctive asparagine, common to all calcitonins, might play a role in the specific processing of calcitonin from its larger parent precursor. It should be noted, however, that secreted forms of calcitonin from a number of species have not been reported to contain carbohydrate (2). Therefore, it is unlikely that this tripeptide sequence represents the only glycosylation site in procalcitonin, and that an additional glycosylation site will be found in the calcitonin precursor when the complete structures of the cryptic peptide regions of this precursor are determined. It is tempting to speculate that the multiple molecular weight species of circulating, immunoreactive calcitonin, observed by a number of investigators (40-42) might represent precursor forms of the hormone which are glycosylated to variable extents.
Processing of secretory proteins, including the initial glycosylation and cleavage of leader sequences, is believed to occur co-translationally on nascent polypeptide chains. Whether the two events occur simultaneously or consecutively is not known. Most likely, cleavage and glycosylation are not obligatorily coupled. In this regard, it appears that the two processes, cleavage and glycosylation, can be dissociated under cell-free conditions. We reported previously the results of the cell-free translations of these identical mRNAs used in the present experiments but using a different preparation of microsomal membranes (4). In those experiments, we found only a product of M , = 12,000 and none of the larger glycosylated product of M, = 17,000. Although obtained from different dogs, the two preparations of microsomal membranes used in these two sets of experiments were prepared from pancreases in an identical manner. The reason for these differences in the processing activities of these two preparations of membranes is unclear, but it is known that different preparations of microsomal membranes differ in glycosylation activity.' The difference in processing activities of our membranes allowed functional separation of cleavage and glycosylation.
Calcitonin represents one of a growing number of small polypeptide hormones for which substantially larger biosynthetic precursors have been identified. These include the M , When the amino acid sequences of these relatively large precursors are determined, it will be of great interest to see whether other biologically active peptides are contained within the sequences analogous to the common large precursor of ACTH, MSH, and the endorphins (9, 43). One intriguing question is how do the cells accurately and faithfully process these precursors to the mature forms of the hormones. It was suggested that glycosylation of biosynthetic precursors, along with the primary structure of the precursors, might play an important role in the determination of the specificity of posttranslational cleavages (9,44). It is notable that the precursor of calcitonin, as well as the precursors of ACTH and vasopressin, contain carbohydrate. It is possible that addition and removal of oligosaccharides from these molecules during their migration through the secretory pathway of the cell influence the patterns and specificities of post-translational processing. The finding of a glycosylated precursor of calcitonin provides further incentive for a systematic analysis of the processing of this hormone in intact cells.