Sep 4, 1990 - To investigate the biological role of variants of hu- man insulin-like growth factor I1 (IGF-11), an extended form designated IGF-IIEZ1, with a ...
Vol. 266, No. 17, Issue of June 15, pp. 11058-11062.1991 Printed in U.S.A.
THEJOUHNALOF BIOLOGICAL CHEMISTRY IC’
1991 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of an Extended Form of Recombinant Human Insulin-like Growth Factor 11” (Received for publication, September 4, 1990)
Bjorn Hammarberg& Michael Tallye, Elisabet SamuelssonS, Henrik Wadenstenn,Erik Holmgrenll, Maris Hartmanisll,Kerstin Halls, Mathias UhlenSII, and Tomas MoksS From the $Department of Biochemistry and Biotechnology, T h e Royal Institute of Technology, S-100 44 Stockholm, Sweden, the §Department of Endocrinology, Karolinska Institute,S-104 01 Stockholm, Sweden, and TlKabiGen AB, Strandbergsgatan49, S - 112 87 Stockholm, Sweden
To investigate the biological role of variants of human insulin-like growth factor I1 (IGF-11),an extended form designated IGF-IIEZ1,with a molecular mass of 9.8 kDa, was produced in Escherichia coli as a stable and soluble secreted fusion protein. After site-specific cleavage of the affinity purified fusion protein, followed by purification using ion exchange and reversed phase chromatography, it could be demonstrated that IGF-IIEsl and IGF-I1 have similar or identical activities according to radioimmunoassayand radioreceptor assay. However, IGF-IIEZ1showed only 1% growth promotion activity as compared with IGF-I1 in a clonal expansion assay using human K562 cells which lacks IGF-I receptors. These results suggest that this extended variant of IGF-I1 can bind to the receptor but has limited growth promoting activity.
kDa form. The second variant has Ser-29 substituted with Arg-Leu-Pro-Gly, but has noE peptide extension (8,9). The Ser-33 substitution is not located at aknown intron/exon hinge region, suggesting that there is more than one gene for IGF-I1 present in the human genome. However, the Ser-29 substitution is located in an intronlexon hinge region, suggesting that an alternativesplicing occurs. The actionof IGF-I1 is mediated by specific receptors, type I and 11. The typeI receptor has higher affinity to IGF-I than IGF-I1 and only moderate affinity to insulin. The type I1 receptor only bind to IGF-I1 (10). This receptor is identical tocationindependentmannose6-phospate receptor (11) which is involved in the transport mechanism of lysosomal enzymesfrom the cell surface and Golgi apparatus to the lysosomes. The type I1 receptor has a single IGF-I1 binding site and shows no homology to the typeI receptor which may explain the difference in affinity. The role of IGF-I1 is not yet fully understood. It has been mayithave The major form of human IGF-11’ found in serum is a 67- suggested from studies in fetal and adult rats that amino acid polypeptide with molecular mass of 7.5 kDa (1). a special role in fetal development, since the IGF-I1 mRNA Different variants and larger forms also exists in serum (2, level in fetal ratis 20-100 times higher than in adult rat (12). 3), brain tissue (4), and cerebrospinal fluid (5),but they are Also fibroblast cultured from fetal tissue release IGF-I1 into from adult tissue release only present in small quantities (5). IGF-I1 is expressed as a the medium, whereas those cultured level is pre-pro formwith an approximate molecular mass of 20.1 IGF-I(13). However, inhumanstheadultserum kDa shown by cDNA (6) and genomic cloning (7). At the higher than earlier in life (14, 15). IGF-I1 may also have an amino-terminal end there is asignal peptide of24 amino effect in the central nerve system (4). IGF-11, but not IGF-I, acids, followed by 67 amino acidwhich upon proteolytic has been shown to promote growth in two human cell lines, processing at Arg-68 give the major IGF-I1 form of 7.5 kDa. the human erythroleukemiacell line K562 (16) and the human This consists of a B, C, and A peptide homologous to pro- T-cell leukemia cell line Jurkat (17). The biological role of the extended formsof IGF-I1 having insulin followed by the nonhomologous D peptide (6residues). whole or parts of the E peptide is even less clear. The question On the extreme carboxyl-terminal end there is an 89-amino acid extension,the E peptide, which has severalpossible arises whether this carboxyl-terminal peptide has a structural processing sites giving rise to themultiplicity of IGF-11s with function in the folding and/or secretory pathway during biodifferent size and charge. synthesis or whether it hasa biological function with different Two variants of human IGF-I1 have been characterized in binding and/or growth promoting properties as compared to more detail. The first variant has a substitution of Cys-Gly- the major 7.5-kDa form. Asp for Ser-33, as well as an carboxyl-terminal extensionby To allow for a more detailed analysisof these nonabundant the first 2 1 amino acids of the E peptide (2) resulting ina 10- variants of IGF-I1 and to investigate the effects of a carboxylterminalextension ingeneral, we decided to produce an *This investigation was supported by grants from the Swedish extended version of IGF-I1 by recombinant techniques. Here, Board of TechnicalDevelopment,the Swedish NaturalResearch Council, and theSwedish Medical Research Council Grants 6897 and we present the cloning, expression, purification, and characterization of an IGF-I1 containing the first 2 1 amino acid of 4224. The costsof publication of this articlewere defrayed in part by the 89-residue-long E peptide as a carboxyl-terminal extenthe payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 sion after the major 67-amino acid form. This formof IGF-I1 solely to indicate this fact. is designated IGF-IIEZ1. 11 To whom correspondence should be addressed. Tel.: 46-8-79087-57; Fax: 46-8-723-18-90. ’ The abbreviations used are: IGF, insulin-like growth factor; Ac, acetate;PAGE, polyacrylamide gel electrophoresis,RIA,radioimmunoassay; RRA, radioreceptor assay; SDS, sodium dodecyl sulfate.
MATERIAL ANDMETHODS
BacterialStrains,Phage,andPlasmid-Escherichia coli strains HBlOl (18) and UT5600 (19) were used for cloning and expression studies. The expression plasmid was pRIT19 (20). Sequencing was
11058
IGF-IIE21 performed using the dideoxy method on single-stranded template obtained by subcloning DNA fragments into the replicative form of M13 phage(21). DNA Construction-Restriction enzvmes and T4-lip.ase (Pharma. cia, Sweden) were used according to the suppliers recommendation. A 2 asdescribed by Maniatis et al. (22). DNA techniques were performed A synthetic gene encoding the E peptide was assembled from four overlappingoligonucleotides synthesized as described earlier(20). Insertion of this synthetic gene into the expression vector pRIT19 resulted in the plasmid pRIT40. Cell Growth-E. coli UT5600 containing the plasmid pRIT40 was., grown overnightin baffled shakeflaskscontaining richmedium, 5 g/liter (Difco), tryptic soy broth, 30 g/liter (Difco) and yeast extract, supplemented with ampicillin, 100 mg/liter. Optical density of the broth was measured a t 600 nm. Protein Cleavage and Purification-Cells were separated by centrifugation 10 min a t 7000 X g, and the fusion protein ZZ-IGF-IIE?I was recovered from the clarified medium and purified on an IgGSepharose fast flow column (Pharmacia, Sweden), as described previously (23). The unique methionine residue between the IGF-IIE,, moiety and the proteinA affinity handle was used for cleavage with 200 times molar excess of CNBr in 70% formic acid as described earlier (24). Thecleavage mixture was lyophilized and redissolved in 30% 1-propanoland purified on a Mono S column(Pharmacia, Sweden) using a gradient from 40 mM NH,Ac, pH 5.2, to 1000 mM NH,Ac, pH 5.5, in 30% 1-propanol. The active fraction was further purified on a reverse phase column, Kromasil KR 150, 5 pm, C-8 (Eka Nobel, Sweden) on a Hewlett-Packard 1090 M HPLC at 40 "C. The IGF-IIE,, was eluted by a gradient between 25 and 55% acetonitrile in 0.1% trifluoroacetic acid. Protein Analysis and BiologicalActiuity-The approximate protein concentrations were determined spectrophotometrically a t 280 nm, using the following extinction coefficients (ml/mg): 0.37 (ZZ-IGFIIE?,),0.17 (ZZ), and 0.49 (IGF-IIE21). The exact measurements and amino acid composition analysis was performed using a Beckman 6300 Amino Acid Analyzer equipped with a System Gold data handling system (Beckman). After hydrolysis with 6 M HC1 at 155 "C for 45 min, the sampleswere analyzed on an ion exchange column with ninhydrin detection. The immunological activity was determined by RIA, the receptor binding activityby a RRA, and growth stimulation by clonal expansion assay of K562 cells that lack the IGF-I receptor, as describedpreviously(10, 16). The active IGF-IIE?I was further analyzed by automaticNH?-terminalsequencingusing a peptide sequencer (Pro sequencer, Milligen) and by '"'Cf plasma desorption mass spectrometer on a Bio-ion 20 (Bio-Ion Nordic AB, Sweden) by the procedure described by H$gset et al. (25). Isoelectric focusing was carried out with precast Ampholine PAG plates, pH 3.5-9.5 (Pharmacia-LKB, Sweden). Before staining with Coomassie Brilliant Blue, the proteins were fixed with 2.5% glutaric dialdehyde followed by 20% trichloroacetic acid for 15 min each. SDS-PAGEwas performed essentially asdescribed by Laemmli (26). II
11059
A 67
rE
AlaLysSer Glu A r g A s p Val Ser T h r Pro Pro Thr Val Leu AI+ -+ CG GCT A A A TCT GAA CGT GAC GTT TCT ACC CCG CCG ACC GTT CTG CGA TTT AGA CTT GCA CTG CAA AGA TGG GGC GGC TGG CAA GAC 81+
Pro
Asp Asn
PhePro
Ar9 Tyr
Pro Val
88 Gly Lys
***
CCA GAC AAC TTC CCG CGT TAC CCA GTT GGT AAA TAA TA GGT CTG TTG AAG GGC GCA ATG GGT CAA CCA TTT ATT ATT CGA 82+
B m
t
M
e
t
]
q
pRIT40
FIG. 1. A, the nucleotide sequence of the gene encoding the last 4 amino acids of the major IGF-I1 and the first21 amino acids of IGFI1 E peptide. The start of each of the four oligonucleotides used to assemble the gene are indicated (AI, A2, B I , and B2) as well as the deduced amino acid sequence. The last amino acids in the major IGF(88)are marked aswell as the startof the I1 (67) and the Epl-peptide E,, peptide. B, schematic drawing of the fusion proteins ZZ-IGF-I1 (pRIT19) and ZZ-IGF-IIE21 (pRIT40) with its methionine residues marked. S represents the signal peptide and 2 represents one IgG binding domain.
12 M34
.m
RESULTS
DNA Construction and Expression in E.coli-A gene fusion system based on the staphylococcal protein A was used for the expression in E. coli (27). The nucleotide sequence encoding the first 21 amino acids of the E peptide of IGF-I1 was synthesized based on the sequence reported by Zumstein et FIG. 2. SDS-PAGE of ZZ-IGF-IIE21fusion protein before al. (2) and assembled as described in Fig. 1A.In order toavoid and after site specific cleavage with CNBr, under nonreducHpaI restriction sites within the synthetic gene fragment, two ing conditions (lanes 1 and 2) and reducing conditions (lanes silent mutationswere introduced. The Ezl gene fragment was 3 and 4 ) . Lanes I and 3, affinity purified fusion protein. Lanes 2 and 4 , after site-specific cleavage. M, marker proteins with the apcloned into the pRIT19 fusionvector using the HpaI site situated at the fifthcodon before the stop codon of the IGF- proximate sizes of 93, 66,43, 31, 21, and 14 kDa. I1 gene and the Hind111 site after the coding region. The recovered and used for purification of ZZ-IGF-IIEzl by IgG resulting fusion vector pRIT40 encodes the signal sequence of staphylococcal protein A (S), two synthetic IgG-binding affinity chromatography. Theproduction level of fusion prodomains (ZZ), a unique methionine residue used for CNBr tein was approximately 30 mg/liter. cleavage, followed by the 88 amino acidresidues of IGF-IIEz~ Analysis of the Affinity Purified IGF-IIEzl Fusion Proteinnonreducing and reducing conditions, schematically shown in Fig. 1B.To circumvent the problems SDS-PAGE, using both of degradation observed for IGF-I1(24), mainly due to prote- shows that also the ompT-negative strain gives a sub-population of degraded protein (Fig. 2, lanes I and 3 ) .Most of the olytic cleavage of a dibasic region by the outer membrane degradation products correspond insize to the ZZ part of 14 protein T (OmpT) (19), an mutant strain lacking this protease can beseen especially was used for the expression. Culture medium from overnight kDa, but other proteolytic productsalso sensitive sites cultures of E. coli UT5600 ( o m p T ) containing pRIT40 was under reducing conditions, indicating that other
IGF-IIEZI
11060
occurwithin the fusion protein. The gel revealed a major fusion protein band at an apparentmolecular mass of 28 kDa. This is larger than the predicted 24 kDa, but it is consistent with whatwas observed previously for ZZ-IGF-I and ZZ-IGF11. The high molecular mass bands under nonreducing conditions (>35 kDa)disappeareduponreduction,suggesting that intermolecular disulfides are responsiblefor the multimer formation. Purification of the Active IGF-IIEzl-The affinity purified fusion protein was treated with CNBr for site-specific cleavage at the unique methionineresidue between the protein A moiety and the recombinant IGF-IIEZI. Analysis by SDSPAGE at nonreducing (Fig. 2, lune 2 ) and reducing (Fig. 2, lane 4 ) conditions show that the 28-kDa band disappears during cleavage and the expected bands,14 kDa (ZZ) and 9.8
Time
B 1 2 3 4 5 6M78910111213
adid
FIG. 3. A , chromatogram a t 280 nm of ion exchange chromatography on a Mono S column of the site-specific cleaved fusion protein. Peaks analyzed by SDS-PAGE are marked with lane numbers. Shadm:ed peak represents the RRA active IGF-IIE,,. Ammonium acetate gradient is indicated, 40 mM to 1 M. R, SDS-PAGE of relevant fractions from the chromatography.Equal amount according toOD,,,, is used in all lanes. Recombinant human IGF-I, molecular mass 7.5 kDa, commercially available from KabiGen AB, is used as a marker in lane 7. The samples were applied using reducing (lanes 1-7) or nonreducing conditions (lanes 9-13). Lanes 1 and 8, mixture after CNHr treatment,but before ion exchangechromatography. M, marker proteins with the approximate sizes of 93,66, 43, 31, 21, and 14 kDa.
kDa(IGF-IIE,,),areenriched.Inaddition, a degradation pattern with lower molecular mass bands could be observed. The cleavage products were lyophilized, redissolved in30% 1propanol, and purifiedby ion exchange chromatography ona Mono S column. A chromatogram a t 280 nm from the purification is shown in Fig. 3A. The different peaks were collected, lyophilized, redissolvedin loading buffer, and analyzed by SDS-PAGE (Fig. 3 R ) using both reducing (lunes 1-7) and nonreducing conditions(lunes 8-13).As a reference, the cleavage mixture before application on theion exchange column is shown (lunes 1 and 8 ) . Only small amounts of multimeric of total protein forms canbe seen in lune 8, due to the amount applied. The first and the major peak (lunes 2 and 9 ) consisted mainly of ZZ, which is consistent with the observed PI for the proteins. The small double peak (lunes 3 and 10) primarily consists of remaining full-length fusion protein ZZ-IGF-IIE,l with an apparent molecular mass of 28 kDa,butalso of degradation products as well as IGF-IIE?,. The minor peak corresponding to lunes 4 and 1 1 shows under nonreducing conditions (lune I l ) , a bandcorrespondingtoIGF-IIErI. Under reducing conditions(lune 4 ) , approximately 50% of the protein appears as twolow molecular mass bands. This indicates that this peak contains a degradation product of IGFIIEZl held together by disulfide bridges that falls apart upon reduction. The shadowed peak (lunes 5 and 12) mainly consists of full-length monomeric IGF-IIE,,. However, a t nonreducing conditions (lune IZ),a larger form corresponding in size to a dimer of the fusion protein appears, with an apparent molecular mass of 56 kDa. Reduction generates a band corresponding to the monomer of ZZ-IGF-IIErl (lune 5 ) .The last peakinthechromatogram (lunes 6 and 13) containsthe multimeric formsof the fusion protein and parts thereof which are washed out from the columna t high salt concentrations. These forms dissociates upon reduction (lune 6 ) and bands corresponding to ZZ-IGF-IIErl,a degradation product at approximately 19 kDa and IGF-IIEZIa t 9.8 kDa appears. Biochemical Characterization-The mainIGF-IIEnlfraction, shadowed in the chromatogram in Fig. 3A, was further purified by reverse phase chromatography. A distinct peak was obtained, which was recovered (shadowed in Fig. 4) and used foranalysis by amino acid content (Fig. 4), NHz-terminal sequencing (Table I), and mass spectroscopy (Table I). In these biochemical characterizations,recombinant IGF-I1 without the E peptide (24) was used as a comparison. The aminoacidanalysisshow a good correlation between the theoreticalvaluesandthemeasuredamino acid content. However, a more precise way to show that the purified recombinant IGF-IIE,, represented the intact molecule is to use
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FIG. 4. Chromatogram at 220 nm of reverse phase chromatography of the main IGF-HEal ion exchange fraction (Fig. 3. A and B. lune 12).The active oeak is indicated. and the amino analysis peak of this
is included.
IGF- IIE21 mass spectrometry. A molecular mass of 9789 daltons could be calculated from the spectrum compared to the theoretical molecular mass of 9807 daltons. This is in good correspondence within the 0.2% error of the method in this molecular mass range. Similar investigationof recombinant IGF-I1gave 7464 and 7471 daltons for measured and theoreticalmolecular mass, respectively. The intact NH2-terminal sequence was confirmed giving 16 readable and identical residues for both IGF-IIE2, and IGF-11. The isoelectric point was determined 8.3, shows a dramatic by isoelectric focusingand the result, PI shift to basic PI. The PI determined for IGF-11, 6.4, is nearly identical with thevalue 6.5 reported by Zumstein et al. (2). Analysis of Biological Activity-The reverse phase chromatography purified IGF-IIEZ1 preparation was diluted according to the proteinconcentrationobtainedintheamino acid analysis. The immunoreactivity of recombinant IGF-I1 and recombinant IGF-IIEZ1was evaluated by competitive inhibiTABLEI Biochemical comparison of recombinant IGF-II and recombinant IGF-IIE,, Molecular Molecular NH,-terminal mass PI mass read/Id deduced measured IGF-IIE,I IGF-I1
9807
9789
8.3
7471
7464 55/55
6.4
FIG. 5. Immunological and biological activity of IGF-I1 and IGFIIEz1.A and B , competitive inhibition of the binding of "'I-IGF-I1 to IGF-IIspecific antibodies raised in hen with a RIA ( A ) or a RRA ( B ) using human placental membranes,by unlabeled IGFI1 (0) and IGF-IIEpl (0).Samples were diluted according to concentrationobtained by determination of amino acid composition. C, RRA for IGF-I, IGF-11, and IGF-IIE,,. Competitive inhibition of the binding of "'I-IGF-I to human placentalmembranes byunlabeled IGF-I (A),IGF-I1 (O), and IGF-IIE,, ( 0 ) .D , growth stimulationof K562 cells by IGFI1 andIGF-IIE,,.Growthstimulation was evaluated as colony formation (>5 cells) of K562 cells in semi-solid agar in the absence or presence of increasing concentrations of IGF-I1 (0) and IGFIIE,] ( 0 )or 100 ng/ml IGF-IIE,, mixed with increasing concentrations of IGF-I1 (A), compared with 10 and 0.1% fetal calf serum ( F C S ) . E , competitive inhibition of bindingtorecombinant ','IIGF-I1 to K562 cells by unlabeled IGFI1 (0) and IGF-IIE,I ( 0 ) .Error bars ( A , H , and D ) indicate the standard deviation of quadruple determination of the samples. No standard deviation in C and I.: was calculated asthesamples only were in triplets.
16/16
11061
tion of binding of ""I-IGF-I1 to specific antibodies, as shown in Fig. 5A. The two preparations disclose almost equipotent inhibition with similarslopes. The antibodiesused here show 0.1-0.5% cross-reactivity with IGF-I. Receptor binding activity for the IGF-I1 and IGF-IIE2] preparations, using human placental membranes,was evaluated by competitive inhibition of the binding of '2sI-IGF-II. In this system, displacement curves show that thereceptor affinities are identicalbetween IGF-I1 and IGF-IIE21 (Fig. 5 B ) . The potency of IGF-IIE21was also similar to IGF-I1 in displacing '"I-IGF-I bound to placenta membrane, and both were about 10-20% as potent as IGF-I (Fig. 5C). Thus, thespecific activity for receptor binding of IGF-IIEyl, as determinedby RRA, is the same as for IGF-11. The growth-promoting activity of IGF-IIE21 ascompared with IGF-I1was studied using the erythroleukemiacells K562, which are unique in binding IGF-I1 but not IGF-I. clonal The growth of these cells are stimulated by IGF-I1 but not IGF-I (10). As seen in Fig. 5 0 , IGF-IIE,, caused a dose-dependent stimulation of clonal growth of K562 cells but was only 1% as potent asIGF-11. When 100 ng/ml IGF-IIEZIwas added to the dose-response curve of IGF-11, the dose-dependent stimulation by IGF-I1 was eliminated and only an effect identical to 100 ng/ml IGF-IIE,' itself was observed. As illustrated in Fig. 5E the IGF-IIEZIwas equipotent to IGF-I1in displacing '"I-IGF-I1 bound to thesecells.
100
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. "
i
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250
300
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100
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100
IGF-IIE21
11062 DISCUSSION
Acknowledgments-We are grateful to Drs. Lars Abrahmsen and Bjorn Nilsson for critical comments and fruitful discussions during this work and to AnetteElmblad for DNA work, Kristina Zachrisson for amino acid analysis, Per Person for mass spectrometry analysis, and Berit Rydlander for biological assays.
The several IGF-11s isolatedinhumansemphasizesthe importance to investigate the different properties of these molecules. One such variant,purified from serum, has totally 90 amino acid residues, with a substitution off Cys-Gly-Arg REFERENCES for Ser-33 to anda 21-amino acid COOH-terminal extension 1. Rinderknecht, E., and Humbel, R. E. (1978) FEBS Lett. 81,283(2). In order to distinguish the effects of these two separate 286 changes and to facilitate the comparison to the major 672. Zumstein, P. P., Luthi, C., and Humbel, R. E. (1985) Proc. Natl. amino acid form, a recombinant IGF-I1was constructed with Acad. Sci. U. S. A. 82, 3169-3172 the carboxyl-terminal extension added to the 67 residues of 3. Gowan, L. K., Hampton, B., Hill, D. J., Schleuter, R. J., and major form. Although, exactly this variant has not yet been Perdue, J. F. (1987) Endocrinology 121, 449-458 4. Haselbacher, G. K., Schwab, M. E., Pasi, A., and Humbel, R. E. isolated from serum, the structural and functional effect of (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 2153-2157 adding the carboxyl-terminal E peptide to an active peptide 5. Haselbacher,. G.,, and Humbel, R. E. (1982) Endocrinology 110, hormone can thusbe investigated. 1822-1824 As already been observed for IGF-I (28) and IGF-I1 (24), 6. Bell. G. I.. Merrvweather. J. P.. Sanchez-Pescador. R.. Stemuien. the yield of active IGF-IIE21 aftersite-specific cleavage of the M: M., 'Priesdey, L., Scott, J., and Rall, L. B. '(1984) Nature affinity purifiedfusion proteinis relatively low. Approxi310, 775-777 7. Dull, T. J., Gray, A,, Hayflick, J. S., and Ullrich, A. (1984) Nature mately 10% of the IGF molecules can be recovered in an 310, 777-781 active form calculated fromthe total amount of fusion protein. 8. Jansen, M., van Schaik, F. M. A,, van Tol, H., Van den Brande, A more detailed characterization revealed that the loss of J. L., and Sussenbach, J. S. (1985) FEBS Lett. 179, 243-246 activity is mainly due to the formation of multimeric forms 9. Hampton, B., Burgess, W . H., Marshak, D. R., Cullen, K. J., and of IGF-IIEZ1fusion protein as well as the degradationof the Perdue, J. F. (1989) J Biol. Chem. 264, 19155-19160 fusion protein by host-specific proteases.' However, the pu- 10. Tally, M., Enberg, G., Li, C. H., and Hall, K., (1987) Biochem. Biophys. Res. Commun. 147, 1206-1212 rified IGF-IIEZ1 obtained after affinity purification, site-specific cleavage, ion exchange chromatography, and preparative 11. Kiess, W., Blickenstaff,G. D., Sklar, M. M., Thomas, C. L., Nissley, S. P., and Sahagian, G. G. (1988) J . Bid. Chem. 263, reverse phase HPLC had the same nearly or identical immu9339-9344 nological (Fig. 5A) and receptor binding activity(Fig. 5 B ) as 12. Moses, A. C., Nissley, S. P., Short, P.A., Rechler, M. M., White, the recombinant 67-amino acid form of IGF-11. The amino R. M., Knight, A. B., and Higa, 0. Z. (1980) Proc. Natl. Acad. Sci. U. S. A . 77,3649-3653 acid analysis, NH2-terminal sequencing and mass spectroscopy confirmed that thepurified molecules had the expected 13. Adams, S. O., Nissley, S. P., Handweger, S., and Rechler, M. M. (1983) Nature 302, 150-153 structure. Interestingly, the basic amino acidsof the E peptide 14. Bennett, A., Wilson, D.M., Liu, F., Nagashima, R., Rosenfeld, shifts the isoelectric point of this IGF-I1 variant to 8.3 as R. G., and Hintz, R. L. (1983) J. Clin. Endocrinol.Metabol. 57, compared with 6.4 for the major IGF-I1 form (Table I). This 609-612 indicates that theE peptide is exposed on the surface,which 15. Enberg, G., and Hall, K. (1984) Acta Endocrinol. 107, 164-170 is consistent with observations of the present three dimen- 16. Tally, M., Li, C. H., and Hall, K. (1987) Biochem. Biophys. Res. Commun. 148,811-816 sional model for IGF-I1 (29), based on theknown structure of 17. Tally, M.(1989) Insulin-likeGrowth Factor ZI. Ph.Dthesis, proinsulin, where the D peptideispointingoutfromthe Karolinska Institute, Stockholm, Sweden molecule. Thus a further carboxyl-terminal extension by the 18. Boyer H. W., and Roulland-Dussoix, D. (1969) J. Mol. Biol. 41, E peptide would be exposed on the surface. 459-472 19. Grodberg, J., and Dunn, J . 5.11988) J . Bacteriol. 170,1245-1253 The IGF-IIE21 variant equipotent to IGF-I1 in the radioimmunoassay was also equipotent to IGF-I1 in radio receptor 20. Hammarberg, B., Nygren, P-A,, Holmgren, E., Elmblad, A., Tally, M., Hellman, U., Moks, T., and Uhlen, M. (1989) Proc. Natl. assay for IGF-I1 using either K562 cells or placenta memAcad. Sci. U. S. A. 86,4367- 4371 branes as matrix. In addition it was equipotent to IGF-I1 in 21. Vieira, J., and Messing, J. (1982) Gene (Amst.) 19, 269-276 the radio receptor assay for IGF-I. Surprisingly thebiological 22. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular effect evaluated asclonal growth of the human erythroleukeCloning: A Laboratory Manual,Cold Spring HarborLaboratory, Cold Spring Harbor, NY ,, mia cells K562, which respond to IGF-I1 but not IGF-I, was reduced to about 1% of that for IGF-11. Furthermore IGF- 23. Moks, T., Abrahmsen, L., Osterlof, B., Josephson, S., Ostling, M., Enfors, S.-O., Person, I., Nilsson, B., andUhlen, M. (1987) IIESladded to the dose-dependentcurve of IGF-I1 blocked its BioTechnology 5,379-382 effect. Since IGF-IIEZIby itself had a weak dose-dependent 24. Hammarberg, B., Moks, T., Tally, M., Elmblad, A., Holmgren, action, it is unlikely that the inhibition of IGF-I1 effect can E., Murby, M., Nilsson, B., Josephson, S., and Uhler,M. (1990) J. Biotechnol. 14, 423- 438 be attributed tosome contamination in preparation, and it is suggested that IGF-IIE21could act as an antagonist. Further 25. Hfgset, A,, Blingsmo, A. R., Saether, O., Gautvik, V. T., Holmgren, E.,Hartmanis, M., Josephson, S., Gabrielsen, 0. S., studies are needed to determine the mechanism of the IGFGordeladze, J. O., Alestrfm, P., and Gautvik, K. M. (1990) J . IIEZ1 binding to insulin-related receptors and to clarify the Biol. Chem. 265,7338-7344 discrepancy found between the receptor bindingof IGF-IIEZ1 26. Laemmli, U. K. (1970) Nature 227, 680-685 27. Nilsson, B., Moks, T., Jansson, B., Abrahmsen, L., Elmblad, A., and thebiological response in targetcells for IGF-11. Holmgren, E., Henrichson, C., Jones, T. A,, and Uhlin, M. This paper describes the first recombinant production of (1987) Protein Eng. 1, 107-113 a n extended form of IGF-11. The recombinant IGF-IIEZ1will 28. Moks, T., Abrahmsen, L., Holmgren, E., Bilich, M., Olsson, A., be valuable for further in vitro studies to explain how larger Uhlen, M., Pohl, G., Sterky, C., Hultberg, H., Josephson, S., forms of IGF-I1 interact with binding proteins and receptors Holmgren. A,, Jornwall.. H.,. andNilsson B. (1987) Biochemistry and what function theyhave. 26, 5239-5244 "
' B. Hammarberg and T. Moks, unpublished data.
29. Blundell, T . L., Bedarkar, S., Rinderknecht, E., and Humbel, R. E. (1978) Proc. Natl. Acad. Sci. U. S. A . 75, 180-184
