©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutational Analysis of the Insulin-like Growth Factor I Prohormone Processing Site (*)

(Received for publication, March 15, 1995; and in revised form, May 2, 1995)

Stephen J. Duguay (1) (2)(§), Jie Lai-Zhang (1), Donald F. Steiner (1) (2)

From the  (1)Department of Biochemistry and Molecular Biology and the (2)Howard Hughes Medical Institute, University of Chicago, Chicago, Illinois 60637

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Insulin-like growth factor I (IGF-I) is a mitogenic peptide that is produced in most tissues and cell lines and plays an important role in embryonic development and postnatal growth. IGF-I is initially synthesized as a prohormone precursor that is converted to mature IGF-I by endoproteolytic removal of the carboxyl-terminal E-domain. Regulation of the conversion of proIGF-I to mature IGF-I is a potential mechanism by which the biological activity of this growth factor might be modulated. Endoproteolysis of the IGF-I prohormone occurs at the unique pentabasic motif Lys-X-X-Lys-X-X-Arg-X-X-Arg-X-X-Arg. Recently, a family of enzymes which cleave prohormone precursors at sites containing multiple basic residues has been discovered. The goals of this study were 1) to determine which basic residues in the pentabasic proIGF-I processing site were necessary for proper cleavage and 2) to examine the role that subtilisin-related proprotein convertase 1 (SPC1/furin) might play in proIGF-I processing. We have shown that an expression vector coding for an epitope-tagged proIGF-I directs synthesis and secretion of mature IGF-I-(1-70), extended IGF-I-(1-76), proIGF-I, and N-glycosylated proIGF-I in human embryonic kidney 293 cells. Extended IGF-I-(1-76) is produced by cleavage at Arg and requires both Arg (P4) and Arg (P1). Cleavage at Arg does not occur in the SPC1-deficient cell lines RPE.40 and LoVo, suggesting that processing at this site is mediated by SPC1. Mature IGF-I-(1-70) is produced by cleavage at Arg and requires both Lys (P4) and Arg (P1). Lys in the P7 position is important for efficient cleavage. SPC1 is not required for processing at Arg since this cleavage occurs in RPE.40 and LoVo cells. These data suggest the existence of a processing enzyme which is specific for the Lys-X-X-Arg motif of proIGF-I.


INTRODUCTION

Insulin-like growth factor I (IGF-I)()circulates in human serum as a 70-amino-acid peptide consisting of four domains: B, C, A, and D. The physiological importance of IGF-I for normal development and growth is underscored by the severity of the phenotype of mice lacking a functional IGF-I gene. Mice carrying a disrupted IGF-I gene display growth deficiencies, delayed bone development, infertility, and a high mortality rate(1, 2) . Liver is the main site of production of circulating IGF-I, although this growth factor is also synthesized and secreted by most tissues. Mature IGF-I can be derived from either of two IGF-I prohormones by removal of the E-domain (see Fig. 2). Alternative splicing of IGF-I mRNA is responsible for generating the two IGF-I prohormones, and the physiological significance of the different forms is unknown(3) . ProIGF-IA contains a 35-amino-acid E-domain, while the E-domain of proIGF-IB is 77 amino acids in length. The sequences of human proIGF-IA and proIGF-IB are identical through the first 16 residues of the E-domain(4) , including the unique pentabasic prohormone cleavage motif Lys-X-X-Lys-X-X-Arg-X-X-Arg-X-X-Arg (Fig. 1). This motif has been conserved in mammals, birds, amphibians, and teleosts.


Figure 2: The proIGF-I-FLAG expression construct. The 24 nucleotides coding for the 8-amino-acid FLAG peptide were inserted between the codons for Ala and Gly of the preproIGF-I cDNA, and the resulting recombinant PCR product was cloned into the pCMV6c expression vector. The CMV promoter and coding region for preproIGF-I-FLAG are boxed. SP, signal peptide; FLAG, FLAG peptide; B, C, A, D, and E refer to the respective domains of proIGF-I. Numbering above the box indicates the position of amino acid residues relative to the first amino acid of the B-domain. Cleavage sites for signal peptidase and the proIGF-I convertase are indicated by vertical arrows. The R71X mutant was generated by converting the codon for Arg to a stop codon. The monoclonal antibody M1 will recognize the FLAG peptide only when it is located at the amino terminus of a protein while the M2 monoclonal antibody will recognize the FLAG peptide in any location. Polyclonal antisera UB3-189 was generated using mature human IGF-I as antigen.




Figure 1: The IGF-I prohormone processing site. The amino acid sequence of the proIGF-I processing site is shown in single-letter code. Basic residues are indicated by boldface type and numbered. The scissile bond connecting the P1 Arg and the P1` Ser is indicated by the vertical arrow.



It is now well established that most peptide hormones and growth factors are initially synthesized as biologically inactive precursors that are converted to active forms by endoproteolysis at specific sites. Proinsulin has served as a model for studies of prohormone processing. From these studies it has been determined that the C-peptide of proinsulin is excised by proteolytic cleavage at paired dibasic residues to produce insulin(5, 6) . Cleavage is mediated by two subtilisin-related proprotein converting (SPC) enzymes, SPC2 (PC2) and SPC3 (PC1/3)(7, 8) . These enzymes are serine proteases and have been shown to process several prohormones to mature hormones, including proglucagon (9) and the proopiomelanocortin precursor(10, 11, 12) . Expression of SPC2 and SPC3 is limited to endocrine and neuroendocrine tissues, and the preferred cleavage site is carboxyl-terminal to Arg-Arg and Lys-Arg doublets(6) . Other members of the mammalian subtilisin-related proprotein convertase family include SPC1 (furin), SPC4 (PACE4), SPC5 (PC4), and SPC6 (PC5/6). SPC1 and SPC4 are widely distributed and cleave proprotein precursors at tri- and tetrabasic sites(13) . The preferred cleavage site for SPC1 appears to be Arg-X-Lys/Arg-Arg(14) , and the Arg-X-X-Arg sequence may serve as a minimal processing site(15) . SPC4 has been reported to have a more strict requirement for a basic residue in the P2 position than does SPC1(16, 17) .

The paradigm established for processing of proinsulin suggests that proIGF-I would be converted to mature IGF-I by cleavage at the carboxyl terminus of Arg, followed by removal of the basic residue by a carboxypeptidase. SPC1 is a candidate proIGF-I convertase since, like IGF-I, it is expressed ubiquitously and it does not have a strict requirement for a basic residue in the P2 position. We have used site-directed mutagenesis to determine the importance of each of the basic residues in the proIGF-I cleavage site for recognition by the processing enzyme. We have also examined the role of SPC1 in proIGF-I processing through the use of cell lines deficient in SPC1 activity.


EXPERIMENTAL PROCEDURES

Expression Constructs

PreproIGF-IA was amplified by PCR from cDNA obtained from the human fibroblast cell line GM 03652C (ATCC). Primers specific for the Met region of the signal peptide (hIGF1-5) and the translation termination codon region of the E-domain (hIGF1-6) were used: 5`-GGGAATTCTTGAAGGTGAAGATGCACAC and 5`-GGGGATCCCCTACATCCTGTAGTTCTTGT. The PCR product was cloned into the EcoRI and BamHI sites of the pCMV6c expression vector, which contains the CMV promotor and the SV40 poly(A) tail, to generate pCMVigf1. pCMVigf1 was then used as a template for recombinant PCR (18) to generate pCMVigf1-FLAG. The signal peptide of IGF-I was amplified using primers hIGF1-5 and Flg-2 (5`-CTTGTTCATCGTCGTCCTTGTAGTCAGCCGTGGCAGAGCTGGT). Flg-2 codes for Thr to Ala of the signal peptide and contains the 24-nucleotide antisense FLAG sequence on the 5` end. The coding region of proIGF-I was amplified using primers hIGF1-6 and Flg-1 (5`-GACTACAAGGACGACGATGACAAGGGACCGGAGACGCTCTGC). Flg-1 codes for Gly to Cys of the B-domain and contains the 24-nucleotide sense FLAG sequence on the 5` end. The PCR products were purified by polyacrylamide gel electrophoresis, and 50 ng of each was mixed, annealed, and extended under PCR conditions and amplified by 12 cycles of PCR with hIGF1-5 and hIGF1-6. The recombinant PCR product was cloned into the EcoRI and BamHI sites of pCMV6c to generate pCMVigf1-FLAG. The fidelity of PCR amplification was verified by DNA sequencing.

Site-directed Mutagenesis

Site-directed mutants were generated using the Altered Sites II in vitro Mutagenesis System (Promega) and double-stranded proIGF-I-FLAG plasmid DNA as template. Oligonucleotide primer sequences and corresponding templates are shown in Table 1. Mutants were subcloned from pAlter to pCMV6c and verified by DNA sequencing.



Cell Culture and Transfections

Human embryonic kidney 293 cells were grown in DMEM (Life Technologies, Inc) with 10% fetal calf serum and 100 units/ml penicillin and 100 µg/ml streptomycin. 293 cells were transfected by the calcium phosphate method(19) . CHO-K1 and RPE.40 cells (provided by Dr. Thomas Moehring, University of Vermont) were grown in Ham's F-12 medium with 5% fetal calf serum and 100 units/ml penicillin and 100 µg/ml streptomycin. CHO-K1 and RPE.40 cells were transfected using LipofectAMINE following the manufacturer's protocol (Life Technologies, Inc).

Cell Labeling, Immunoprecipitation, and Gel Electrophoresis

24 h after transfection cells were washed twice in phosphate-buffered saline and incubated for 2 h in cysteine-free (or histidine-free) Dulbecco's modified Eagle's medium with 1 mg/ml bovine serum albumin, 100 units/ml penicillin, and 100 µg/ml streptomycin. The medium was then replaced with cysteine-free (or histidine-free) Dulbecco's modified Eagle's medium containing 100 µCi/ml [S]cysteine (Amersham, 1000 Ci/mmol) or [C]histidine (DuPont NEN, 385 Ci/mmol), 1 mg/ml bovine serum albumin, 100 units/ml penicillin, and 100 µg/ml streptomycin. After labeling for 24 h, conditioned medium was collected and centrifuged to remove cellular debris. Conditioned medium was stored at -20 °C. Cells were washed twice with phosphate-buffered saline and removed from the plate in immunoprecipitation (IP) buffer (25 mM Tris-Cl, pH 7.4, 300 mM NaCl, 1 mM CaCl, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100), sonicated, and stored at -20 °C.

Conditioned medium (1 to 5 ml) was acidified with trifluoroacetic acid (0.1% final concentration) and loaded onto a Sep-Pak C-18 cartridge (Millipore) that had previously been equilibrated with 20% acetonitrile, 0.1% trifluoroacetic acid. After washing the column in 20% acetonitrile, 0.1% trifluoroacetic acid, peptides were eluted in 80% acetonitrile, 0.1% trifluoroacetic acid, lyophilized, and reconstituted in 500 µl of IP buffer. Immunoprecipitation was performed with 3 µg of M1 or M2 anti-FLAG monoclonal antibody (Kodak/IBI) or 5 µl of UB3-189 anti-human somatomedin-C polyclonal antisera (National Hormone Pituitary Program, NIDDK) overnight at 4 °C. M1 and M2 immune complexes were precipitated with protein G-Sepharose (Pharmacia Biotech Inc.), and UB3-189 immune complexes were precipitated with protein A-Sepharose. The immunoprecipitates were washed twice in buffer containing 25 mM Tris-Cl, pH 7.4, 300 mM NaCl, 1 mM CaCl, and then once in buffer consisting of 25 mM Tris-Cl, pH 7.4, 140 mM NaCl, 1 mM CaCl. Cell lysates were thawed and centrifuged to remove cell debris. 500 µl of supernatant was preabsorbed with protein G-Sepharose and immunoprecipitated with M1 antibody as described above.

Immunoprecipitates were solubilized in SDS-sample buffer containing 2-mercaptoethanol and denatured by heating at 95 °C for 5 min. Samples were electrophoresed on Tricine-buffered gels with a 4% stack and a resolving gel of 10%, 16.5%, or 20% polyacrylamide(20) . Gels were fixed in 10% acetic acid, 25% isopropyl alcohol for 30 min, and then soaked in a fluorographic solution (Amplify, Amersham) for 15 min. Dried gels were exposed to x-ray film (Kodak) with an intensifying screen.

Deglycosylation

7 ml of conditioned medium from [S]cysteine-labeled cells was concentrated on a Sep-Pak and lyophilized as described above. The lyophilized proteins were solubilized in 0.1% SDS, denatured by heating at 95 °C for 10 min, and placed on ice. 77 µl of 10 deglycosylation buffer (200 mM NaHPO, pH 7.2, 5% Nonidet P-40) was added, and 100-µl aliquots were incubated with 2 milliunits of neuraminidase (sialidase), 2.5 milliunits of O-glycosidase, 0.4 unit of N-glycosidase F (Boehringer Mannheim), or combinations of these enzymes for 24 h at 37 °C. After incubation, 400 µl of IP buffer was added, and epitope-tagged peptides were immunoprecipitated with M1 antibody and run on 10% polyacrylamide gels as described above.


RESULTS

The FLAG Peptide Does Not Interfere with Processing of PreproIGF-I

Our initial attempts to study the processing of proIGF-I were hampered by difficulties in immunoprecipitating proIGF-I from conditioned medium. Commercially available antisera to IGF-I have been generated using mature IGF-I as antigen, and the E-domain of proIGF-I may interfere with epitope recognition. To circumvent this problem we designed a preproIGF-I expression vector that contained an epitope tag, the 8-amino-acid FLAG peptide, between Ala of the signal peptide and Gly of the B-domain (Fig. 2). Two monoclonal antibodies to the FLAG peptide have been generated. The M1 antibody requires calcium for binding and will only recognize a FLAG peptide with an exposed amino terminus. The M1 antibody can therefore be used to determine whether the signal peptide has been removed from proIGF-I-FLAG. The M2 antibody will recognize the FLAG peptide in any location and should therefore immunoprecipitate all epitope-tagged peptides. To obtain an accurate molecular weight marker for IGF-I-FLAG, we mutated Arg of proIGF-I-FLAG to a stop codon. This R71X mutant directs synthesis of the 70-amino-acid mature IGF-I molecule with the FLAG peptide at the amino terminus ( Fig. 2and Table 1).

Before proceeding with biosynthetic studies, it was necessary to determine if the presence of the FLAG peptide would alter processing of the IGF-I prohormone. 293 cells were transfected with IGF-I, R71X, and IGF-I-FLAG expression vectors, and [S]cysteine-labeled proteins were immunoprecipitated from conditioned medium using UB3-189 anti-human IGF-I antisera (Fig. 3). Two proteins of 7-8 kDa were immunoprecipitated from conditioned media of cells transfected with CMVigf1. Two proteins were also immunoprecipitated from conditioned media of cells transfected with CMVigf1-FLAG. These peptides have a molecular mass of 9-10 kDa and represent the epitope-tagged homologues of the IGF-I peptides synthesized from the CMVigf1 vector. The 9-kDa band generated from the IGF-I-FLAG transfection migrates at the same position as the R71X mutant, which has a predicted molecular mass of 8640 daltons, indicating that proIGF-I-FLAG is processed at Arg. The larger molecular mass bands of the IGF-I and IGF-I-FLAG doublets may represent carboxyl-extended forms of mature IGF-I.


Figure 3: The FLAG peptide does not interfere with proIGF-I processing. 293 cells were transfected with IGF-I, R71X, or IGF-I-FLAG expression vectors, and [S]cysteine-labeled IGF peptides were immunoprecipitated using the UB3-189 antisera and resolved on Tricine-buffered 16.5% polyacrylamide gels, as described under ``Experimental Procedures.'' M indicates mock transfection. The molecular masses of the C-methylated protein markers are indicated.



When conditioned medium from R71X-transfected 293 cells was immunoprecipitated with M1, M2, and UB3-189 antisera, a predominant band was seen at 9 kDa (Fig. 4). The slightly larger band recognized by the M1 antibody may be an oxidized form of IGF-I. When conditioned medium from IGF-I-FLAG-transfected 293 cells was immunoprecipitated with these antisera, several peptides were visualized. The 9-kDa band generated from the R71X transfections was also present in IGF-I-FLAG-transfected media and was recognized by anti-FLAG and anti-human IGF-I antisera, indicating that this band represents mature IGF-I-FLAG. The 10-kDa band from the IGF-I-FLAG-transfected media was also recognized by all three antisera and may represent a carboxyl-extended form of mature IGF-I-FLAG. In addition to the 9-10-kDa doublet, the M1 and M2 antibodies immunoprecipitated peptides of 14 kDa and 16 kDa, as well as a broad band from 19-21 kDa. Since these peptides were recognized by both M1 and M2, they do not represent forms of IGF-I containing a signal peptide and thus the FLAG peptide does not interfere with signal peptidase activity. The higher molecular weight bands, which were not immunoprecipitated with the UB3-189 antisera, may be forms of proIGF-I.


Figure 4: PreproIGF-I-FLAG is processed to mature IGF-I-FLAG. 293 cells were transfected with R71X or IGF-I-FLAG expression vectors, and [S]cysteine-labeled peptides were immunoprecipitated from aliquots of reconstituted conditioned medium with M1, M2, or UB3-189 antisera. Immunoprecipitates were resolved on Tricine-buffered 16.5% polyacrylamide gels, as described under ``Experimental Procedures.'' The molecular masses of the C-methylated protein markers are indicated.



ProIGF-I and N-Glycosylated ProIGF-I Are Secreted by Transfected 293 Cells

Selective amino acid labeling was employed in order to determine whether the high molecular weight proteins immunoprecipitated from IGF-I-FLAG-transfected conditioned media by anti-FLAG antibodies were unprocessed or partially processed proIGF-I molecules. Human proIGF-IA contains two histidine residues, both of which are located in the E-domain. Therefore, histidine labeling should be specific for proIGF-I as opposed to oligomers or aberrantly processed mature IGF-I. Fig. 5shows the results of [S]cysteine and [C]histidine labeling of cells transfected with the IGF-I-FLAG expression vector. The 9-10-kDa doublet migrated as a single band on this 10% polyacrylamide gel and was clearly visible when cells were labeled with [S]cysteine but was not present when labeled with [C]histidine. In contrast, 14-kDa and 19-21-kDa proteins were visible from both [S]cysteine and [C]histidine labeling, and the [C]histidine-labeled 16-kDa band could be seen after overexposure (data not shown).


Figure 5: Multiple forms of unprocessed proIGF-I are secreted. 293 cells were transfected with the IGF-I-FLAG expression vector and metabolically labeled with [S]cysteine (Cys) or [C]histidine (His). Peptides were immunoprecipitated from reconstituted conditioned medium with the M2 antibody and resolved on Tricine-buffered 10% polyacrylamide gels. M = mock, T = transfected. The molecular masses of C-methylated protein markers are indicated.



The predicted molecular mass of proIGF-I-FLAG is 12.7 kDa, in close agreement with the 14-kDa band seen in [C]histidine labeling. The proIGF-IA E-domain contains one potential N-glycosylation site at Asn and several serine and threonine residues which could be used for O-glycosylation. To determine if the 16-kDa and 19-21-kDa peptides were glycosylated forms of proIGF-I, peptides from conditioned medium of proIGF-I-FLAG-transfected 293 cells were subjected to treatment with various glycosidases. As shown in Fig. 6, neuraminidase and O-glycosidase had no effect on the electrophoretic mobility of proIGF-I peptides. However, the 19-21-kDa band completely disappeared after digestion with N-glycosidase while the intensity of the 14-kDa and 16-kDa peptides increased dramatically. It is therefore likely that the 19-21-kDa peptide is N-glycosylated proIGF-I-FLAG, the 14-kDa peptide is proIGF-I-FLAG, and the 16-kDa peptide may be partially glycosylated proIGF-I-FLAG.


Figure 6: ProIGF-I is N-glycosylated. 293 cells were transfected with the IGF-I-FLAG expression vector and metabolically labeled with [S]cysteine. Conditioned medium was concentrated on a Sep-Pak, lyophilized, reconstituted, and denatured. One-tenth volume of 10 deglycosylation buffer was then added, and aliquots were incubated in the absence of enzyme(-), or with neuraminidase (S), O-glycosidase (O), N-glycosidase (N) alone or in combinations for 24 h at 37 °C. Peptides were immunoprecipitated with M1 antibody and run on Tricine-buffered 10% polyacrylamide gels. The molecular masses of the C-methylated protein markers are indicated.



Conversion of ProIGF-I to Mature IGF-I Occurs Intracellularly

Since there are examples of growth factors that can be proteolytically activated intra- or extracellularly, it was of interest to determine the location of conversion of proIGF-I to mature IGF-I. Cell lysates from R71X or IGF-I-FLAG-transfected 293 cells were immunoprecipitated with the M1 antibody, and peptides were resolved on 16.5% polyacrylamide gels. The 9-10-kDa processed IGF-I-FLAG doublet, as well as proIGF-I-FLAG and glycosylated proIGF-I-FLAG, were clearly visible in cell lysates, indicating that conversion can occur intracellularly (Fig. 7). We have also demonstrated that proIGF-I-FLAG peptides are not processed to mature IGF-I-FLAG when incubated with conditioned media from 293 cells or 50% fetal calf, rat, or mouse serum for up to 24 h at 37 °C. However, degradation of proIGF-I-FLAG occurs when incubated with trypsin (data not shown).


Figure 7: Conversion of proIGF-I to mature IGF-I occurs intracellularly. 293 cells were transfected with the R71X or IGF-I-FLAG expression vector, and [S]cysteine-labeled peptides were immunoprecipitated from cell lysates with the M1 antibody and resolved on Tricine-buffered 16.5% polyacrylamide gels, as described under ``Experimental Procedures.'' M indicates mock transfection. The molecular masses of the C-methylated protein markers are indicated.



Processing of ProIGF-I Mutants in 293 Cells

To determine the importance of each of the basic residues in the pentabasic proIGF-I processing motif for prohormone conversion activity, we systematically mutated each basic residue to an uncharged residue (Table 1). Single, double, and triple mutants were generated. Since our initial experiments with the R71X and IGF-I-FLAG expression vectors indicated that proIGF-I was being cleaved at Arg and a downstream site, we also made two additional stop codon mutants for use as molecular weight markers. Arg and Arg were mutated to stop codons, generating R74X and R77X, respectively. In several cases, substitution of a basic residue with an uncharged amino acid changed the electrophoretic mobility of the protein. In some of these instances it was necessary to make additional stop codon mutants in order to correctly identify the processing site.

Fig. 8A shows the results of expression of the single substitution mutants in 293 cells. On Tricine-buffered 20% polyacrylamide gels, the R71X mutant migrates as a single band. The R74X mutant migrates as a poorly resolved doublet, and the R77X mutant migrates as a clearly defined doublet. This gel electrophoresis system is therefore capable of resolving IGF-I-FLAG peptides of 78, 81, and 84 amino acids in length. It is also interesting to note that both the R74X and R77X peptides are processed at Arg, indicating that as few as two residues on the carboxyl terminus of Arg are sufficient for processing activity.


Figure 8: Processing of proIGF-I mutants in 293 cells. 293 cells were transfected with expression constructs coding for wild-type (WT) and mutated proIGF-I-FLAG, as indicated: panel A, R71X, R74X, R77X, WT, K68G, R71A, R74A, R77A; panel B, K65A/R71X, K65A, K65A/R74A, K65A/R77A; panel C, K65A/K68G/R71X, K65A/K68G; panel D, R77X, K68G/R71A, K68G/R74A, K68G/R77A, R71A/R77A, R74A/R77A. Cells were metabolically labeled with [S]cysteine, and peptides were immunoprecipitated with the M1 antibody. Immunoprecipitates were resolved on Tricine-buffered 20% polyacrylamide gels, as described under ``Experimental Procedures.'' The molecular masses of the C-methylated protein markers are indicated.



As shown previously ( Fig. 3and Fig. 4), wild-type IGF-I-FLAG is processed at two sites yielding a peptide that migrates with the R71X standard at 9 kDa as well as a 10-kDa peptide. Comparison of these peptides with the pattern produced by expression of R74X and R77X indicates that the 10-kDa IGF-I-FLAG peptide is generated by cleavage at Arg (Fig. 8A). In several different transfection experiments, we have observed that approximately 50% of proIGF-I-FLAG is cleaved at Arg and 50% at Arg. We have not observed evidence for cleavage at Arg (Table 2).



Mutation of Arg to alanine (R71A) abolishes processing at this site, indicating that the basic residue is required at the P1 site. Processing at Arg is not qualitatively affected (Fig. 8A). Mutations at either Arg (R74A) or Arg (R77A) eliminate processing at Arg without qualitatively affecting cleavage at Arg (Fig. 8A).

The K68G mutant processing pattern is qualitatively similar to that of wild-type proIGF-I processing except that all bands migrate slightly faster on Tricine-buffered gels (Fig. 8A). This band shift may be attributed to size and/or charge differences of the lysine and glycine side chains. When K68G and K68G/R71X peptides are run together on Tricine-buffered 20% gels, it is clear that processing does not occur at Arg. The K68G peptide migrates more slowly than the K68G/R71X stop codon mutant (data not shown) and is thus processed at a downstream site such as Arg. The P4 lysine residue is therefore required for processing at Arg.

When Lys is mutated to Ala (K65A), the peptide is processed at two sites (Fig. 8B). The smaller band migrates with the K65A/R71X mutant, indicating that it is generated by cleavage at Arg. Since the larger band of the doublet is well resolved, it is likely that it represents processing at Arg.

Three K65A double mutants were generated. The K65A/R74A and K65A/R77A mutants migrate as a single band with the same molecular weight as the K65A/R71X mutant (Fig. 8B). These mutants are therefore cleaved at Arg but not Arg. The processed K65A/K68G mutant has a higher molecular weight than the corresponding K65A/K68G/R71X mutant, indicating that the two upstream lysine residues are required for cleavage at Arg (Fig. 8C). It is likely that cleavage of K65A/K68G occurs at Arg since the K65A/K68G and K65A/K68G/R71X bands are clearly resolved.

While it was not possible to definitively assign cleavage locations for the K68G double mutants, it is clear that these mutants are processed very poorly, if at all, at Arg. Only 10% of the K68G/R74A mutants and 16% of the K68G/R77A mutants were processed (Fig. 8D and Table 2). 79% of the K68G/R71A mutant was processed, and it is likely that cleavage occurred at Arg since this site is preferred to Arg in proIGF-I-FLAG. When Arg is isolated by substitution of alanine for Arg and Arg (R71A/R77A), only 8% of the precursor is cleaved. When Arg and Arg are changed to alanine residues (R74A/R77A), approximately 50% of the precursor is cleaved at Arg (Fig. 8D and Table 2).

Processing of ProIGF-I in RPE.40 Cells

The data on processing of proIGF-I mutants indicated that processing at Arg was prevented in all mutants having a substitution at either Arg or Arg ( Fig. 8and Table 2), suggesting that this sequence might be a cleavage site for SPC1, which has minimally been defined as RXXR(15) . These data also demonstrate that processing at Arg requires both Lys (P4) and Arg (P1). It is not yet clear if SPC1 will process precursors containing a Lys for Arg substitution in the P4 position. In order to determine if SPC1 is involved in proIGF-I processing, we expressed IGF-I-FLAG in CHO-K1 and RPE.40 cells. RPE.40 cells were derived from CHO.K1 cells exposed to Pseudomonas exotoxin A (PEA) and selected for resistance (21) . RPE.40 cells fail to process precursor membrane glycoproteins of several viruses, as well as the insulin proreceptor. This processing deficiency can be corrected by transfection with SPC1 cDNA(22, 23) .

When CHO-K1 cells were transfected with the R71X and R77X stop codon mutants, peptides corresponding to cleavage products at Arg and Arg were immunoprecipitated from conditioned media (Fig. 9). Transfection with IGF-I-FLAG produced the Arg and Arg doublet, as well as proIGF-I and glycosylated proIGF-I. Expression of R71X and R77X in RPE.40 cells resulted in the same pattern of immunoprecipitable peptides as was seen in transfected CHO-K1 cells. However, when IGF-I-FLAG was expressed in RPE.40 cells, a single band of mature IGF-I, corresponding to IGF-I-FLAG processed at Arg, was observed. This indicates that RPE.40 cells process proIGF-I at Arg but not Arg. We have also observed a single band corresponding to Arg cleavage when IGF-I-FLAG was transfected into LoVo cells (data not shown). LoVo cells do not process the MET protooncogene or the insulin proreceptor(24) , and this processing deficiency has been attributed to a defective SPC1 gene (25) .


Figure 9: Processing of proIGF-I in RPE.40 cells. CHO-K1 and RPE.40 cells were transfected with R71X, R77X, or IGF-I-FLAG expression vectors and cells were metabolically labeled with [S]cysteine. Peptides were immunoprecipitated with the M1 antibody and resolved on Tricine-buffered 16.5% polyacrylamide gels, as described under ``Experimental Procedures.'' M indicates mock transfection. The molecular masses of the C-methylated protein markers are indicated.




DISCUSSION

We have used the pCMVigf1-FLAG expression vector and human embryonic kidney 293 cells to study proteolytic processing of human proIGF-I. Using the UB3-189 anti-human IGF-I antisera, we have shown that the pCMVigf1 and pCMVigf1-FLAG expression vectors direct synthesis and secretion of processed IGF-I and IGF-I-FLAG, respectively (Fig. 3). Using the M1 and M2 monoclonal antibodies, we have demonstrated that the FLAG epitope does not interfere with removal of the signal peptide (Fig. 4). PreproIGF-I-FLAG is therefore faithfully processed to mature IGF-I-FLAG and can be used to study post-translation processing of proIGF-I.

When expressed in 293 cells, pCMVigf1-FLAG directs synthesis of multiple forms of high molecular weight proIGF-I-FLAG peptides which are converted to proIGF-I-FLAG by incubation with N-glycosidase ( Fig. 5and Fig. 6). Although glycosylated forms of human proIGF-I have not been described previously, Simmons et al.(26) have shown that rat proIGF-I can be N-glycosylated in an in vitro translation system. Interestingly, peptides generated from initiation of translation at Met or Met, but not Met, were substrates for N-glycosylation. The pCMVigf1-FLAG expression vector contains translation initiation codons at Met and Met, but Met has been eliminated. N-Glycosylated proIGF-I has not been identified in vivo, and the significance of this post-translational modification is unknown. However, it has been demonstrated that glycosylation can affect proteolytic processing of some precursor proteins. For instance, N-glycosylation of the human influenza virus hemagglutinin precursor at Asn can inhibit processing at Arg(27) .

Proprotein precursors can be converted to mature forms intracellularly, at the cell surface membrane, or extracellularly. Pulse-chase experiments have shown that the E-domain of proIGF-II is cleaved at several sites during transport through the cell. Most of these cleavages, including the final cleavage to produce the 7-kDa mature IGF-II, appear to occur very late in the secretory pathway and may also occur extracellularly(28) . We detect mature IGF-I-FLAG in 293 cell lysates, indicating that conversion can occur intracellularly (Fig. 7). We have not seen evidence for processing of proIGF-I-FLAG to mature IGF-I-FLAG when proIGF-I-FLAG peptides were incubated with conditioned media from 293 cells or serum from mouse, rat, or fetal calves (data not shown).

Expression of wild-type and mutant proIGF-I-FLAG constructs in 293 cells indicates that the IGF-I prohormone can be cleaved at two sites, Arg and Arg. The Arg cleavage site conforms to the minimal SPC1 cleavage site defined by Molloy et al.(15) with Arg as the P4 residue and Arg as the P1 residue. All mutants containing alanine in place of Arg or Arg eliminate processing at Arg (R74A, R77A, K65A/R74A, K65A/R77A, K68G/R77A, R71A/R77A, R74A/R77A). In all mutants containing substitutions of nonbasic residues for Lys, Lys, or Arg, the percentage of precursor processing at Arg is increased (K65A, K68G, R71A, K65A/K68G, K68G/R71A) (Table 2). One explanation for the increased processing at Arg in these mutants is that the secondary structure of the peptide may have been modified in a way that allows for more favorable enzyme-substrate interactions. However, each of the Lys, Lys, and Arg mutants also decreases or eliminates processing at Arg. It is therefore likely that the increased processing at Arg is a result of the creation of a less efficient Arg cleavage site. In addition to mutational analysis, data obtained from the expression of wild-type proIGF-I-FLAG in SPC1-deficient cell lines also implicate SPC1 as the Arg cleavage enzyme. When wild-type proIGF-I-FLAG is expressed in CHO-K1 cells, cleavage occurs at Arg and Arg. However, when expressed in RPE.40 cells, a CHO-K1 derivative lacking SPC1 activity, cleavage of proIGF-I-FLAG occurs at Arg but not Arg (Fig. 9). Processing at Arg, but not Arg, was also observed when proIGF-I-FLAG was expressed in the SPC1-deficient LoVo cell line (data not shown). It therefore appears that SPC1 is required for cleavage of proIGF-I-FLAG at Arg but not Arg.

Since IGF-I isolated from human serum is 70 amino acids long, it is likely that final maturation occurs by cleavage of the precursor at Arg. The presence of a second cleavage site at Arg, which affects processing efficiency at Arg, makes it difficult to determine the minimal processing motif necessary for final maturation. Mutational analysis has revealed that Lys and Arg are both essential for processing at Arg. Substitution of glycine for Lys (K68G) or alanine for Arg (R71A) eliminates processing at Arg (Fig. 8A, Table 2). The P7 lysine residue may also be important for efficient processing since 28% of the K65A mutant is cleaved at Arg, as opposed to 55% for wild-type proIGF-I. It thus seems paradoxical that the K65A/R74A and K65A/R77A double mutants are processed more efficiently at Arg than the K65A single mutant (68% and 92%, respectively) (Table 2). A likely explanation, however, is that the double mutants eliminate the P4 and P1 residues, respectively, of the SPC1 cleavage site, therefore making more substrate available for the Arg cleaving enzyme. Arg alone is not sufficient for processing since the K68G/R74A mutant is cleaved very poorly (Fig. 8D, Table 2).

The optimal SPC1 cleavage site has been defined as RXK/RR (29) . SPC1 will also cleave some precursors with an RXXR motif with reduced efficiency (15, 16, 29) while other precursors with this motif are not cleaved(30) . It is unclear whether SPC1 will cleave substrates containing a lysine residue in the P4 position. The fusion glycoprotein precursor (F) of the Ulster strain of Newcastle disease virus contains the KQGR cleavage motif. The F glycoprotein of Newcastle disease virus strain Ulster was not cleaved when coexpressed or incubated in vitro with SPC1(30) . A virulent avian influenza virus hemagglutinin containing the RKKR cleavage motif was processed efficiently by SPC1 in both coexpression and in vitro incubation experiments. When the P4 arginine of this motif was mutated to lysine to create KKKR, 75% of the hemagglutinin was cleaved when coexpressed with SPC1, and the hemagglutinin was not cleaved when incubated with SPC1 in vitro(31) . Our data on expression of proIGF-I-FLAG in RPE.40 and LoVo cells indicate that SPC1 is not necessary for final maturation of IGF-I. The identity of the proIGF-I converting enzyme is unknown. SPC4 is a candidate because it is expressed in many tissue types. However, this enzyme apparently has a more strict requirement for a basic residue in the P2 position than does SPC1(16, 17) . For many proprotein precursors that are processed at basic residues, the scissile bond is on the carboxyl terminus of the P1 residue. After cleavage of the scissile bond by a specific endoprotease, the basic residues are removed by carboxypeptidases. Recently, a metalloendopeptidase which cleaves peptide bonds on the amino terminus of arginine residues was cloned from a rat testis cDNA library(32) . Although the proIGF-I cleavage site is similar to the motif recognized by enzymes of the SPC family, it is possible that a different class of enzyme may convert proIGF-I to mature IGF-I.

It is interesting that the R74X and R77X stop codon mutants are cleaved at Arg. This indicates that as few as two amino acids on the carboxyl terminus of the P1 residue are sufficient for precursor processing by the enzyme. Processing of these mutants, as well as mutants containing alanine substitutions at Arg or Arg, also demonstrates that cleavage at Arg need not precede cleavage at Arg. Pulse-chase experiments have shown that proIGF-II is cleaved at multiple sites in the E-domain(28) . Similar experiments will be necessary to determine if proIGF-I is cleaved at Arg before final maturation. At this time there is no evidence for the existence of IGF-I-(1-77) in vivo and the SPC1-mediated cleavage observed at Arg in our experiments may be a consequence of overexpression. Nonetheless, this observation is interesting in terms of SPC site selection specificity, and proIGF-I may be a useful model for studying this aspect of prohormone processing. Three repetitive motifs of basic-X-X-basic residues are contained within only 10 amino acids. SPC1 recognizes the motif RXXR. The motif KXXR may also be recognized by SPC1, but it is likely to be cleaved by another converting enzyme as well. The RXXR motif does not appear to be recognized by SPC1 or the proIGF-I converting enzyme. In fact, this site appears to be highly refractory to cleavage. Attempts to force cleavage at Arg by isolating the RXXR (K68G/R77A) or the single Arg (R71A/R77A) were unsuccessful (Fig. 8D, Table 2). Knowledge of the role of nonbasic residues in proIGF-I processing, or the secondary structure of the pentabasic motif, could provide useful insights into processing specificity of prohormone convertases.


FOOTNOTES

*
This work was supported by the Howard Hughes Medical Institute and Grant DK13914 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Howard Hughes Medical Institute, University of Chicago, 5841 S. Maryland Ave., MC 1028, Chicago, IL 60637. Tel.: 312-702-1328; Fax: 312-702-4292.

The abbreviations used are: IGF-I, insulin-like growth factor I; SPC, subtilisin-related proprotein convertase; PCR, polymerase chain reaction; CMV, cytomegalovirus; DMEM, Dulbecco's modified Eagle's medium; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.


ACKNOWLEDGEMENTS

We thank Dr. Graeme Bell for providing the pCMV6c expression vector and Dr. Thomas Moehring for the CHO-K1 and RPE.40 cell lines. The UB3-189 polyclonal antisera was a gift from Dr. Terry Taylor of the National Hormone and Pituitary Program. We also thank Paul Gardner for synthesis of oligonucleotides. We are very grateful to Drs. Shu Jin Chan, Yves Rouillé, and Kaare Lund for many helpful discussions and comments on this manuscript.


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