6
Biochimica et Biophvsica/Iota. 1051 (1990l 6 13 Elsevier
BBAMCR 12588
The E-domain peptide of rat pro-insulin-like growth factor II (prolGF-II)" properties of the peptide in serum and production by rat cell lines V i n c e n t W. H y l k a * a n d D a n i e l S. S t r a u s Division of Biomedical Sciences and Department of Biology, University of California, Riverside, CA (U.S.A.) (Received 13 February 1989) (Revised manuscript received 28 August 1989)
Key words: Insulin like growth factor II; E-domain peptide; (Rat serum); (BRL-3A rat liver cell); (Rat embryo fibroblast)
We previously identified a naturally occurring peptide fragment derived from the carboxyl terminal region of the E-domain of pro-insulin-like growth factor II (prolGF-llttT_lSs) in medium conditioned by cultured BRL-3A rat liver cells. In the present study we utilized a radioimmunoassay (IliA) for this peptide to measure physiological concentrations of the peptide in media and serum. Serum levels of the E-domain peptide were very high in the 5-day neonatal rat and declined thereafter to reach low levels in adult rat serum. Chromatography of adult rat serum on Sephadex G-75 in 1 M acetic acid yielded a single broad peak of E-peptide immunoreactivity that coeluted with a synthetic E-peptide standard. However, chromatography of day 5 neonatal rat serum on Sephadex G-75 yielded two peaks of immunoreactivity. One of the peaks coeluted with a synthetic E-peptide standard, whereas the other peak eluted in a region where higher molecular weight proteins typically elute. Experiments aimed at determining whether adult rat serum contained a binding protein for the E-domain peptide revealed that: (1) serum contains little, if any, binding protein for the E-domain peptide, (2) sermn contains a proteinase activity that degrades the E-domain peptide, and (3) the proteinase activity can be eliminated by acetic acid/ethanol extraction. Of several rat cell lines tested (BRL-3A, rat embryo fibroblasts (REF), hepatoma cell lines (I-14, HTC), GH 3 pituitary tumor cells, and normal rat kidney fibroblasts (NRK)), only BRL-3A and REF cells secreted measurable E-domain peptide into the medium. In addition, it was found that some component(s) of serum could stimulate secretion of E-domain peptide from BRL-3A and REF cells. Chromatography of the immunoreactivity from BRL-3A and REF-conditioned media on Sephadex G-75 in 1 M acetic acid yielded a single peak that coeluted with a synthetic E-domain peptide standard. Since secretion of the E-dmnain peptide parallels the expression of IGF-II, the RIA for the prolGF-lI E-domain peptide may he useful for studies of the biosynthesis and secretion of IGF-II under different physiological conditions. The RIA for the IGF-ll E-domain peptide has two technical advantages over the RIA for IGF-II, namely, the lack of interference by IGF binding proteins and the relative ease with which large quantities of pure antigen can he synthesized.
Introduction Insulin-like growth factor II is a single-chain polypeptide mitogenic hormone that is structurally related to proinsulin [1]. The mitogenic activity of this hormone in cell culture suggests that it has a growth-promoting
* Present address: Livingston Research Center, Department of Obstetrics and Gynecology, University of Southern California Medical Center, 1321 N. Mission Road, Los Angeles, CA 90033, U.S.A. Abbreviation: prolGF-II, pro-insulin-like growth factor II. Correspondence: D.S. Straus, Division of Biomedical Sciences, University of California, Riverside, CA 92521-0121, U.S.A.
function in vivo; however, its exact physiological function is not well understood at present. In the rat, the level of expression of the IGF-II gene [2,3] and concentration of IGF-II in plasma [4,5] are high in the fetus and neonate, but decline to very low levels in the adult. High concentrations of IGF-II have also been found in the fetal and neonatal lamb and in the fetal guinea pig [6,7]. These observations suggest that IGF-II may be a major regulator of fetal growth [7,8]. Other evidence suggests an additional role for IGF-II in the central nervous system. IGF-II gene expression in the brain persists into adulthood in rats [2], and IGF-II and variant forms of IGF-II have been found in brain and cerebrospinal fluid in rats and humans [9-11]. These observations suggest a possible growth-promoting or
0167-4889/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
regulatory role for IGF-II in the central nervous system. IGF-II is synthesized as a prohormone precursor that contains an 89 residue carboxyl terminal extension domain (E-domain) that is cleaved off during processing of the hormone [12,13]. The high degree of sequence conservation (79%) between the E-domain regions of rat and human prolGF-II [12,13] has led to speculation that this region may have a biological function. We reported previously the first identification of a naturally occurring peptide fragment of the E-domain region of rat prolGF-II [14]. This peptide was found in media as a secretory product produced by cultured BRL-3A rat liver cells. The E-domain peptide begins at position 117 of the rat prohormone sequence, and its molecular weight determination [15] is consistent with it being equivalent to the carboxyl terminal 40 amino acids of the pro-IGF-II E-domain. We recently synthesized this peptide and developed a radioimmunoassay for the synthetic peptide [15]. This RIA is analogous to the insulin C-peptide RIA, in that it detects a portion of the IGF-II prohormone that is not represented in mature IGF-II. Using this RIA we previously determined that E-domain peptide immunoreactivity was present at high levels in 5-day-old rat pups, but was very low in adult rats [15]. The present study was undertaken to utilize the RIA for further studies of the developmental regulation of the E-domain peptide in rat serum, and to examine the production of this peptide by cultured rat cell lines. Materials and Methods
Animals 3-month-old Sprague-Dawley-derived rats were obtained from Simonsen-Gilroy (Gilroy, CA). The animals were maintained on a 12 h light, 12 h dark cycle and received water and rat chow ad libitum. Rat pups of 5, 11, 15, 20 and 24 days after birth were obtained after breeding in our vivarium. No parental rats were utilized in these studies. All rats were decapitated and serum was immediately stored at - 7 0 ° C until analyzed. Collection of conditioned medium Stock cultures of all cells were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum, 100/~g streptomycin/ml and 70 U penicillin/ml. For collection of conditioned medium, the BRL-3A rat liver cell line [16], REF rat embryo fibroblasts (prepared from rat embryos on day 14 of gestation), N R K normal rat kidney fibroblasts [17], G H 3 rat pituitary tumor cells [18], H4 rat hepatoma cells [19] and HTC rat hepatoma cells [20], were plated at 400 000 cells per 25 cm 2 flask and cultured in MEM supplemented with 100/xg streptomycin/ml and 70 U penicillin/ml. Conditioned medium was collected from flasks
after 2 days of incubation. At that time, cell number was determined by counting with a Coulter counter.
Sephadex G-75 chromatography Conditioned medium (5-10 ml) from BRL-3A or REF cells, serum (2-18 ml) from neonatal or adult rats, or molecular weight standards were acidified with acetic acid to a final concentration of 1 M acetic acid in 20 ml final volume. The sample was centrifuged at 14 000 x g for 15 min and the resulting supernatant (approx. 20 ml) was applied to a Sephadex G-75 column (2.5 x 86 cm for serum; 2.5 x 90 cm for medium) which was equilibrated in 1 M acetic acid. Fractions of 4.3 ml were collected and aliquots of fractions were lyophilized, reconstituted in RIA buffer (0.01 M sodium phosphate, 0.154 M NaC1, 0.02% sodium azide, 0.1% gelatin, 0.1% bovine serum albumin (RIA grade), and 0.01% Triton X-100, pH 7.6), and neutralized prior to measurement of IR-E-peptide. Neutralized aliquots containing precipitates were clarified by centrifugation prior to assay. Peptides Synthetic rat prolGF-IInT_156 (pro-rlGF-II117_156; = E - d o m a i n peptide), and [Tyrn6]pro-rlGF-IllaT_156 (Tyr n6 analog) were synthesized and purified as previously described [15]. Measurement of E-domain peptide binding proteins in serum To examine whether serum contained a binding protein for pro-rlGF-Ii17_156, 20 /~1 of iodinated Tyr n6 analog ((6-8). 105 cpm; approx. 10 ng) was incubated with 1 ml of serum from an adult female rat pool at 4 ° C for 20 h. After the incubation period, the sample was applied to a Sephadex G-75 column (2.5 x 87 cm) previously equilibrated in 0.03 M sodium phosphate, 0.154 M NaC1, 0.02% sodium azide, 0.1% gelatin (pH 7.4). Fractions were collected (5.7 ml) at 4°C, and 3.0 ml aliquots were counted in a gamma counter. In similar experiments, 125I-Tyrn6 analog was incubated with 0.03 M phosphate buffer (as a control), or with an acetic acid/ethanol extract of the adult rat serum pool (see below for extraction methodology) prior to chromatography on Sephadex G-75. For analysis of radioactivity recovered near the void volume, fractions containing the radioactivity were pooled, lyophilized, reconstituted in 20 ml of 1 M acetic acid, and rechromatographed on a Sephadex G-75 column (2.5 x 90 cm) equilibrated with 1 M acetic acid. Competition experiments were performed as described above, with the addition of 10 ~g of unlabeled Tyr 116 analog. Serum extraction Extractions of serum were performed in order to determine the conditions which would eliminate binding
proteins while retaining maximum E-domain peptide. In a preliminary experiment, both iodinated E-domain peptide and an unlabeled E-domain peptide RIA standard were extracted separately with either 12.5% 2 M HC1/87.5% ethanol as per the methodology of Daughaday et al. [21] or with 15% 8 M acetic acid/85% ethanol as described below. After percent recoveries were determined (via % cpm recovered and % recovery of immunoactivity via RIA), it was determined that the acetic acid/ethanol extraction resulted in a higher percentage recovery (76%) of E-domain peptide immunoactivity compared with HC1/ethanol extraction (30%). Thus the acetic acid/ethanol method was used for all extraction experiments. (The % recovery of 125I-labeled E-peptide was higher in the HCl/ethanol method, but the percentage of immunoactivity was much less (30% compared with the the acetic acid/ethanol procedure, indicating loss of E-domain peptide immunoactivity when HC1 was used for extraction.) Acid/ethanol-extracted serum was prepared by mixing 1 ml of adult female rat serum pool with 4 ml of 15% 8 M acetic acid/85% ethanol in polypropylene tubes. Tubes were mixed and centrifuged at 2000 x g for 30 min at 4 ° C. The supernatant was transferred to another polypropylene tube, frozen at - 7 0 ° C and lyophilized. Lyophilized samples were reconstituted in 1 ml of 0.03 M sodium phosphate, 0.154 M NaC1, 0.1% gelatin, 0.02% sodium azide, 0.0025% phenol red and neutralized just prior to binding studies. Lioer extraction Livers were immediately excised and quick-frozen on dry ice. Extraction was performed at 4 ° C using a Dounce homogenizer in 1 M acetic acid. Aliquots (50-100 /~1) of homogenate were lyophilized and later used for protein analysis. Homogenates were centrifuged at 28 000 x g for 1 h at 4 ° C. Supernatants were decanted, frozen at - 7 0 °C and lyophilized. Lyophilized samples were diluted in RIA buffer containing 0.0025% phenol red. Samples were then neutralized, transferred to microfuge tubes, and clarified by centrifugation at top speed in a microfuge for 30 rain at 4 ° C. Supernatants were then assayed for IR-E-domain peptide. In some cases (to demonstrate parallelism of the extracts to the E-domain peptide standard curve) reconstituted samples were diluted prior to RIA. Measurement of protein Protein content of liver homogenates was determined by the Folin phenol method [22] using bovine serum albumin as standard. Lyophilized homogenate was dissolved in hot 0.1 M NaOH prior to analysis. RIA for the E-domain peptide Immunoreactive E-domain peptide was assayed using a polyclonal antibody raised in rabbits as previously
described [15]. The only modification from this procedure was that the incubation was performed at 4 ° C overnight. Bound ligand was separated from free using protein A (Staphylococcus aureus." IgSorb, The Enzyme Center, Boston, MA) to precipitate bound ligand. Statistics Data were analyzed using analysis of variance followed by Duncan's new multiple range test [23]. These procedures were corrected for unequal sample sizes, where appropriate. The significance level was chosen at P < 0.05. Results
The prolGF-II E-domain peptide that we previously identified and for which we developed a radioimmunoassay [14,15] consists of the carboxyl terminal 40 amino acids of the rat prolGF-II E-domain, beginning at position 117 of the prohormone (see Fig. 1). Data in Fig. 2 indicate that serum E-domain peptide levels were very high on day 5 after birth and decreased sharply between days 5 and 25 ultimately to attain very low levels in the adult. The relationship between the immunoreactive material found in neonatal vs. adult serum was analyzed further by comparison of acid-gel chromatographic profiles of immunoreactive material from adult and neonatal sera (Fig. 3a, b). Chromatography of adult rat serum on Sephadex G-75 in 1 M acetic acid yielded a single major broad peak of E-domain peptide immunoactivity that coeluted with a synthetic E-domain peptide standard (Fig. 3a). However, chromatography of day 5 neonatal rat serum yielded 2 peaks of immunoreactivity (Fig. 3b). One of the two peaks of activity (271 ml eluent) coeluted with a synthetic E-domain peptide standard, whereas the second peak (155 ml eluent) eluted in a region where higher molecular weight proteins typically elute (to the left of the cytochrome c standard). Serial dilutions of liver extracts from day 11 and adult rats were shown to displace tracer in parallel to the displacement seen using synthetic E-domain peptide (data not shown). The profile of the concentration of hepatic E-domain peptide from day 11, 15, 20, 24 and adult rats is shown in Fig. 4. Hepatic content of E-domain peptide decreased significantly after day 11, with very low levels measured in the adult liver. Several experiments were performed to determine whether rat serum contained a binding protein for the E-domain peptide. Results from these experiments are shown in Figs. 5a, b and c. In the first series of experiments (Fig. 5a) neutral-gel filtration chromatography was performed with an 125I-E-domain peptide control sample, and this sample was found to elute as a single major peak of radioactivity at 335 ml eluent. However, when the 125I-E-domain peptide was in-
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Fig. 1. The sequence of rat prolGF-lI predicted from cDNA sequence analysis [12,13]. The N-terminal 67 amino acids correspond to the IGF-II hormone. The prohormone also contains a carboxyl terminal extension of 89 residues (68-156) termed the E-domain. The E-domain peptide (which is underlined in the top portion of the figure) represents the carboxyl terminal 40 residues of the E-domain (positions 117-156). Numbers represent residue position.
cubated with 1 ml adult rat serum for 20 h at 4 °C prior to chromatography, four peaks of radioactivity were observed, including two small peaks near the void volume (peaks A and B), a major peak at 335 ml eluent, and a small peak of radioactivity at 545 ml eluent (peak C). Peaks A and B did not diminish even if the incubation of serum and 125I-E-domain peptide was performed in the presence of 10 #g of unlabeled competing peptide (data not shown). This indicates that these two peaks do not represent E-domain peptide complexed with a specific binding protein.
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Fig. 2. E-domain peptide levels in serum of neonatal and adult rats. Adult rats were 4 m o u t h s old. Values represent the m e a n + S.E. of four or five individual samples. F o r adult, each sample = one rat; for neonates, each sample = a pool of four or five pups.
The two small peaks of radioactivity (peaks A and B) that eluted near the void volume were further chromatographed on Sephadex G-75 under acidic conditions (1 M acetic acid), and the profiles of radioactivity in the the resulting fractions are shown in Fig. 5b. The majority of the radioactivity of both peaks remained in the high molecular weight region following acid-gel filtration chromatography indicating that this material did not dissociate in 1 M acetic acid. This provides further evidence that these two peaks do not represent E-domain peptide complexed with a specific binding protein. An additional series of experiments was directed toward determining whether acetic acid/ethanol extraction of rat serum prior to incubation with 125I-E-domain peptide and subsequent neutral-gel filtration chromatography would eliminate peaks A, B, or C that had been generated after incubation with unextracted serum (see Fig. 5a). The results of neutral-gel chromatography of a sample of 125I-E-domain peptide preincubated with acid/ethanol extracted serum are shown in Fig. 5c. Extraction of serum did not eliminate the small peak of radioactivity seen near the void volume (175 ml eluent). However, extraction of serum prior to incubation totally eliminated the peak of radioactivity at 545 ml eluent (peak C, Fig. 5a). Several rat cell lines were examined for their ability to secrete E-domain peptide in culture (Table I). Of the six cell lines examined (BRL-3A rat liver cells, rat
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ELUENT VOLUME (ml) Fig. 3. Chromatographic profiles of adult and neonatal rat serum on Sephadex G-75 in 1 M acetic acid. Absorbance of a 1 : 10 dilution is shown by the dotted line. Solid line, E-domain peptide, normalized to an 18.85 ml sample of serum. The dimensions of the column were 2.5x90 cm. Fractions of 4.3 ml were collected. Molecular weight standards are: cytochrome c (13 320), ribonuclease A (13 680), insulin (5733), bacitracin (1400) and synthetic E-domain peptide (4432). Vt marks the elution position of NaCI. Although not shown in the figure, the ribonuclease A standard ran at 249 ml. (A) An aliquot of pooled adult female rat serum (18.85 mi) was acidified to 1 M acetic acid and chromatographed using 1 M acetic acid as eluent. Aliquots of fractions were lyophilized and reconstituted in RIA buffer prior to assay. The experiment was performed four times (using different serum pools) with identical results. (B) An aliquot (7.5 ml) of pooled day 5 neonatal rat serum (n = 20) was diluted to 18.85 ml and acidified to 1 M acetic acid. Sample was loaded onto the column and eluted with 1 M acetic acid as described above. E-peptide levels have been normalized for an 18.85 ml serum sample for comparison with Fig. 3a. Experiment was performed twice (using two different serum pools) with identical results.
e m b r y o f i b r o b l a s t s ( R E F ) , H 4 a n d H T C h e p a t o m a cells, G H 3 p i t u i t a r y t u m o r cells, a n d a n o r m a l r a t k i d n e y f i b r o b l a s t i c cell l i n e ( N R K ) ) , o n l y B R L - 3 A l i v e r c e l l s and REF fibroblasts produced measurable quantities of E - d o m a i n p e p t i d e . A l t h o u g h B R L - 3 A cells p r o d u c e d 3-5-times more E-domain peptide than REF cells (P < 0.01), b o t h cell t y p e s e x h i b i t e d i n c r e a s e d p r o d u c t i o n o f t h e E - d o m a i n p e p t i d e ( n o r m a l i z e d p e r cell n u m b e r ) when the concentration of fetal bovine serum was inc r e a s e d f r o m 0.3% t o 1.0% ( P < 0.01). To examine and compare the nature of the immunor e a c t i v i t y o f t h e B R L - 3 A vs. R E F cells, s a m p l e s o f m e d i a c o n d i t i o n e d b y t h e s e cells w e r e c h r o m a t o g r a p h e d o n S e p h a d e x G - 7 5 i n 1 M a c e t i c a c i d ( F i g . 6), a n d
profiles were compared. Acid-gel filtration chromatography of conditioned media from BRL-3A or REF cells yielded a single peak of immunoreactivity that coeluted with a synthetic E-domain pep.tide standard (280 ml eluent).
TABLE I Secretion of immunoreactive E-domain peptide from various rat cell lines in culture
Cells were incubated in minimal essential medium for 2 days prior to collection of media, n.d., not detectable; FCS, fetal calf serum. Rat cell line
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Fig. 4. Analysis of rat liver extracts for content of E-domain peptide. E-domain pcptide levels from extracted livers of neonatal and adult rats. Values represent the mean+S.E, of three or four individual samples. Asterisks indicate significant difference vs. mean values for day 11 values ( P < 0 . 0 5 ) . Mean levels for adult liver were not significantly different compared with levels from livers on day 20.
Values represent the mean+S.E, of three or four independent measurements. * Significantly different ( P < 0 . 0 1 ) from the same cell line in 1% FCS. ** Significantly different ( P < 0.01) from the REF cells at the same percentage of FCS. a
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We report here further studies using an RIA for the rat proIGF-II E-domain peptide aimed at determining the nature of the developmental regulation of this peptide in rat serum, and identifying cultured rat cell lines that might secrete this peptide. Previously, we identified this peptide as a naturally occurring secretory product found in rat serum [15] and in medium conditioned by BRL-3A rat liver cells [14,15]. Serum levels of E-domain peptide decreased 60-fold from the day 5 rat pup to the mature adult, and this decline of E-peptide concentrations during the neonatal period is consistent with earlier results on IGF-II in the rat [4,21]. These
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data indicate that E-domain peptide levels roughly parallel levels of IGF-II in serum. To investigate further the nature of circulating E-domain peptide during the neonatal period, we subjected serum from day 5 pups and adult rats to gel filtration chromatography in acetic acid. Adult rat serum exhibited a single broad peak of immunoactivity. However, the day 5 neonatal serum profile exhibited two peaks of immunoactivity, one that coeluted with a synthetic E-peptide standard, and a second peak that eluted in a region where higher molecular weight proteins elute (Fig. 3b). We can probably eliminate the idea that this
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Fig. 5. Analysis of adult rat serum for E-domain peptide binding proteins. Total recovery of radioactivity from columns ranged from 86-90%. (a) Neutral-gel filtration chromatography on Sephadex G-75 of n~I-E-domain peptide after a 20 h incubation at 4 ° C with: (i) buffer alone (dosed circles) or with 1 ml of normal adult female serum (open circles). The dimensions of the column were 2.5 x 87 cm. Eluent was phosphate buffer (pH 7.4) (see Materials and Methods). Fractions of 5.7 ml were collected. Molecular weight standards were: a-chymotrypsinogen (23240), ribonuclease A (13680) and adenosine triphosphate (551). (b) Acid-gel filtration chromatography of peaks A and B from the experiment shown in a. Peaks from the experiment shown in a were acidified to 1 M acetic acid and re-chromatographed on a 2.5 × 90 cm column of Sephadex G-75 using 1 M acetic acid as eluent. Fractions of 4.3 ml were collected. Open circles represent peak A, and closed circles represent peak B. Molecular weight standards as in Fig. 3. (c) Neutral-gel filtration chromatograpfiy on Sephadex G-75 of nSI-E-domain peptide (closed circles) or 125I-E-domain peptide+ acetic acid/ethanol-extracted adult rat serum (open circles). The dimensions of the column were 2.5 × 87 cm. The column was eluted with phosphate buffer (pH 7.4). Fractions of 5.7 ml were collected. Prior to chromatography, tracer was incubated for 20 h at 4 ° C with either buffer alone or with an acetic a c i d / e t h a n o l extract of 1 ml of adult female rat serum. Molecular weight markers as in a.
12 higher molecular weight material is due to interaction with a binding protein, since the chromatography was performed under acidic conditions (1 M acetic acid). Since the antibody used in the present study is directed against the carboxyl terminus of the proIGF-II E-domain, we suspect that this higher molecular weight material represents the intact proIGF-II molecule. We cannot, however, conclusively rule out the possibility that this material is a large fragment of proIGF-II lacking the amino terminal portion of the proIGF-II molecule. The appearance of the higher molecular weight peak in neonatal serum appears to indicate a difference in processing a n d / o r secretion of proIGF-II in the rat pup as compared with the adult rat. The peak of immunoreactive E-domain peptide obtained with adult rat serum is typically very broad and might conceivably contain a very small amount of high molecular weight material at the extreme leading portion of the peak. However, it is certain that this higher molecular weight material is not present in media conditioned by BRL-3A or REF cells (Fig. 6). The greater relative amount of high molecular weight immunoreactive E-domain material in the neonatal rat as compared with adult rat under acidic chromatographic conditions may indicate: (1) lower activity of IGF-II processing enzymes in rat pup vs. adult rat; (2) over-saturation of the processing enzymes by the large amount of proIGF-II substrate present in rat pup and consequent secretion of the unprocessed hormone; or (3) developmental control over secretion of proIGF-II. In the 5 day neonatal rat, serum E-domain peptide concentrations were very high and declined to low levels in the adult. Hepatic concentrations of E-domain peptide also decreased from high levels in the day 11 neonate to very low levels in the adult rat. The decline in hepatic IGF-II E-domain peptide from neonatal to adult parallels the decline in hepatic IGF-II mRNA observed in other studies [2,3]. We measured immunoreactive E-domain peptide in medium conditioned by various rat cell lines. Although two hepatoma cell lines (H4, HTC) did not secrete measurable E-domain peptide, the BRL-3A liver cells and rat embryo fibroblasts did secrete this peptide. REF cells have also been shown previously to secrete large quantities of IGF-II [24]. Both the BRL-3A and R E F cells were induced to secrete significantly more E-domain peptide on a per cell basis when cultured in medium containing 1% as compared with 0.3% serum. The effect of increased serum on E-domain peptide production was greater with the REFs than with the BRL-3A cells, consistent with the greater serum dependency of the REFs. The stimulatory effect of serum on p r o l G F - I I E-domain peptide production may reflect general metabolic stimulation by serum or may indicate that some component in serum stimulates
E-domain peptide synthesis/secretion from these cells. In regard to the latter possibility, it is of interest to note that Clemmons et al. [25,26] previously observed that platelet-derived growth factor (a component of serum) stimulated the secretion of IGF-I by human fibroblasts. The immunoreactivity of media conditioned by BRL3A or REF cells was shown to be an identical, single chromatographic peak, that coeluted with the synthetic E-domain peptide. The observation that neither intact proIGF-II nor higher molecular weight fragments of the proIGF-II E-domain are detected in conditioned medium using this RIA suggests that proteolytic removal of the carboxyl terminal 40 amino acids of the E-domain (i.e., the E-domain peptide) may be an early step in the processing of proIGF-II in these rat cells. To address the possibility that there might be serum proteins that bind the E-domain peptide, we examined rat serum for evidence of such a protein. Since there is no amino acid sequence homology between IGF-II and the proIGF-II E-domain peptide, it seemed unlikely that IGF-II binding proteins would have affinity for the E-domain peptide. However, it was conceivable that some other protein(s) might bind the E-domain peptide. Overnight incubation of 125I-E-peptide with serum resulted in only a minor shift of radioactivity to the void volume area of the neutral G-75 column (peaks A and B, Fig. 5a). If this shift of activity was due to interaction with a binding protein(s), then acid-gel filtration chromatography should have caused a dissociation of binding protein and tracer, thus resulting in a shift in the elution peak of radioactivity (i.e., 125I-E-domain peptide) back to the position where the synthetic E-domain peptide standard ehited. However, the radioactivity remained near the void volume area after acid-gel chromatography (Fig. 5b), indicating that this minor peak of radioactivity is probably not due to interaction with a binding protein. The radioactivity in the void volume may be an aggregate of tracer, as has been suggested for other peptides [21]. A c i d / e t h a n o l extraction of serum prior to overnight incubation did not eliminate the aggregated material (Fig. 5c, 175 ml eluent). Thus little, if any, binding protein for the E-domain peptide was found in adult rat serum. In contrast, rat serum contains large quantities of IGF-II binding proteins [21]. It was interesting to note that overnight incubation of 125I-E-peptide with serum also generated a radioactive peak that eluted after ATP upon neutral-gel chromatography (peak C, Fig. 5a). That this peak ehited well after ATP (which was used as a low molecular weight standard), suggested that rat serum contained a proteinase that degraded the 12SI-E-domain peptide to a much smaller fragment, and that this degradation product had some affinity for the Sephadex G-75 column. This proteinase was eliminated by extraction of the serum with a c i d / e t h a n o l (compare Fig. 5a with 5c). More experimentation is needed to characterize the
13 n a t u r e of this m i n o r p r o t e i n a s e activity. It is p o s s i b l e that this p r o t e i n a s e activity m a y b e similar to a n acidl a b i l e p r o t e i n a s e activity p r e v i o u s l y d e s c r i b e d in h u m a n s e r u m [27], a l t h o u g h the activity in rat s e r u m a p p e a r s to b e m u c h lower t h a n that p r e v i o u s l y o b s e r v e d in h u m a n serum. Since I G F - I I a n d the E - d o m a i n p e p t i d e are p r o c e s s e d p r o d u c t s o f the s a m e p o l y p e p t i d e p r e c u r s o r molecule, they are synthesized in e q u i m o l a r quantities. O u r s t u d ies to d a t e i n d i c a t e t h a t levels o f the E - d o m a i n p e p t i d e in c u l t u r e m e d i a a n d s e r u m r o u g h l y p a r a l l e l levels of I G F - I I . T h u s the R I A for the E - d o m a i n p e p t i d e will be useful for future studies on the b i o s y n t h e s i s a n d secretion of I G F - I I . This w o u l d be a n a l o g o u s to the R I A for the insulin C - p e p t i d e . R a d i o i m m u n o a s s a y s for the insulin-like g r o w t h factors are p r o b l e m a t i c b e c a u s e of interference b y s e r u m b i n d i n g proteins. This interference is very difficult to e l i m i n a t e c o m p l e t e l y . F o r e x a m p l e , recent studies have d e m o n s t r a t e d that I G F b i n d i n g p r o t e i n s c a n n o t be c o m p l e t e l y r e m o v e d f r o m rat serum [28] o r ovine p l a s m a [29] b y a c i d / e t h a n o l extraction. Thus, the assay for the p r o - I G F - I I E - d o m a i n p e p t i d e has a technical a d v a n t a g e over the R I A for I G F - I I in the lack of i n t e r f e r e n c e b y b i n d i n g proteins. A n a d d i t i o n a l a d v a n t a g e o f the E - d o m a i n p e p t i d e R I A is the relative ease with which large q u a n t i t i e s o f p u r e a n t i g e n can b e synthesized. I n p a r t i c u l a r , the E - d o m a i n p e p t i d e d o e s not c o n t a i n a n y cysteine residues, so that f o l d i n g of the p e p t i d e a n d p r o p e r a l i g n m e n t o f disulfide linkages is n o t a p r o b l e m .
4
5 6 7 8
9 10 11 12 13 14 15 16 17 18 19 20 21
Acknowledgments W e t h a n k N i c h o l e M i h a r a for excellent technical assistance, a n d M a e G o r d o n for her w o r d p r o c e s s i n g skills. This w o r k s u p p o r t e d b y U S D A c o m p e t i t i v e g r a n t 88-37265-4115 a n d N I H g r a n t s R 0 1 - D K 3 9 7 3 9 a n d F32DK-07721.
References 1 Froesch, E.R. and Zapf, J. (1985) Diabetologia 28, 485-493. 2 Brown, A.C., Graham, D.E., Nissley, S.P., Hill, D.J., Strain, A.J. and Rechler, M.M. (1986) J. Biol. Chem. 261, 13144-13150 (and additions: J. Biol. Chem. 262, 2424-2425). 3 Lund, P.K., Moats-Staats, B.M., Hynes, M.A., Simmons, J.G.,
22 23 24 25 26 27 28 29
Jansen, M., D'Ercole, A.J. and Van Wyk, J.J. (1986) J. Biol. Chem. 261, 14539-14544. Moses, A.C., Nissley, S.P., Short, P.A., Rechler, M.M., White, R.M., Knight, A.B. and Higa, O.Z. (1980) Proc. Natl. Acad. Sci. USA 77, 3649-3653. Daughaday, W.H., Parker, K.A., Borowsky, S., Trivedi, B. and Kapadia, M. (1982) Endocrinology 110, 575-581. Gluckman, P.D. and Butler, J.H. (1983) J. Endocrinol. 99, 223-232. Daughaday, W.H., Yanow, C.E. and Kapadia, M. (1986) Endocrinology 199, 490-494. Sara, V.R. and Carlsson-Skwirut, C. (1987) in Highlights on Endocrinology. (Christiansen, C. and Riis, B.J., eds.), pp. 275-280, Bogtrykkeri, Norhaven, A/S. Binoux, M., Hossenlopp, P., Lassarre, C. and Hardouin, N. (1981) FEBS Lett. 124, 178-184. Haselbacher, G. and Humbel, R. (1982) Endocrinology 110, 1822-1824. Haselbacher, G.K., Schwab, M.E., Pasi, A. and Humbel, R.E. (1985) Proc. Natl. Acad. Sci. USA 82, 2153-2157. Dull, T.J., Gray, A., Hayflick, J.S. and Uilrich, A. (1984) Nature (London) 310, 777-781. Whitfield, H.J., Bruni, C.B., Frunzio, R., Terrell, J.E., Nissley, S.P. and Rechler, M.M. (1984) Nature (London) 312, 277-280. Hylka, V.W., Teplow, D.B., Kent, S.B.H. and Straus, D.S. (1985) J. Biol. Chem. 260, 14417-14420. Hylka, V.W., Kent, S.B.H. and Straus, D.S. (1987) Endocrinology 120, 2050-2058. Straus, D.S., Coppock, D.L. and Pang, K.J. (1981) Biochem. Biophys. Res. Commun. 100, 1619-1625. DeLarco, J.E. and Todaro, G.J. (1978) J. Cell Physiol. 94, 335-342. Tashjian, A.H., Jr., Yasumura, Y., Levine, L., Sato, G.H. and Parker, M.L. (1968) Endocrinology 82, 342-352. Straus, D.S. and Takemoto, C. (1987) J. Biol. Chem. 262, 1955-1960. Thompson, E.B., Tomkins, G.M. and Curran, J.F. (1966) Proc. Natl. Acad. Sci. USA 56, 296-303. Daughaday, W.H., Mariz, I.K. and Blethen, S.L. (1980) J. Clin. Endocrinol. Metab. 51,781-788. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Bancroft, T.A. (1968) Topics in Intermediate Statistical Methods. p. 100, Iowa State University Press, Ames, IA. Adams, S.O., Nissley, S.P., Handwerger, S. and Rechler, M.M. (1983) Nature (London) 302, 150-153. Clemmons, D.R., Underwood, L.E. and Van Wyk, J.J. (1981) J. Clin. Invest. 67, 10-19. Clemmons, D.R. (1984) J. Clin. Endocrinol. Metab. 58, 850-856. Powell, D.R., Lee, P.D.K., Chuang, D., Liu, F. and Hintz, R.L. (1987) J. Clin. Endocrinol. Metab. 119, 868-875. Daughaday, W.H., Kapadia, M. and Mariz, I. (1987) J. Lab. Clin. Med. 109, 355-363. Mesiano, S., Young, I.R., Browne, C.A. and Thorburn, G.D. (1988) J. Endocrinol. 119, 453-460.
Biochimica et Biophvsica/Iota. 1051 (1990l 6 13 Elsevier
BBAMCR 12588
The E-domain peptide of rat pro-insulin-like growth factor II (prolGF-II)" properties of the peptide in serum and production by rat cell lines V i n c e n t W. H y l k a * a n d D a n i e l S. S t r a u s Division of Biomedical Sciences and Department of Biology, University of California, Riverside, CA (U.S.A.) (Received 13 February 1989) (Revised manuscript received 28 August 1989)
Key words: Insulin like growth factor II; E-domain peptide; (Rat serum); (BRL-3A rat liver cell); (Rat embryo fibroblast)
We previously identified a naturally occurring peptide fragment derived from the carboxyl terminal region of the E-domain of pro-insulin-like growth factor II (prolGF-llttT_lSs) in medium conditioned by cultured BRL-3A rat liver cells. In the present study we utilized a radioimmunoassay (IliA) for this peptide to measure physiological concentrations of the peptide in media and serum. Serum levels of the E-domain peptide were very high in the 5-day neonatal rat and declined thereafter to reach low levels in adult rat serum. Chromatography of adult rat serum on Sephadex G-75 in 1 M acetic acid yielded a single broad peak of E-peptide immunoreactivity that coeluted with a synthetic E-peptide standard. However, chromatography of day 5 neonatal rat serum on Sephadex G-75 yielded two peaks of immunoreactivity. One of the peaks coeluted with a synthetic E-peptide standard, whereas the other peak eluted in a region where higher molecular weight proteins typically elute. Experiments aimed at determining whether adult rat serum contained a binding protein for the E-domain peptide revealed that: (1) serum contains little, if any, binding protein for the E-domain peptide, (2) sermn contains a proteinase activity that degrades the E-domain peptide, and (3) the proteinase activity can be eliminated by acetic acid/ethanol extraction. Of several rat cell lines tested (BRL-3A, rat embryo fibroblasts (REF), hepatoma cell lines (I-14, HTC), GH 3 pituitary tumor cells, and normal rat kidney fibroblasts (NRK)), only BRL-3A and REF cells secreted measurable E-domain peptide into the medium. In addition, it was found that some component(s) of serum could stimulate secretion of E-domain peptide from BRL-3A and REF cells. Chromatography of the immunoreactivity from BRL-3A and REF-conditioned media on Sephadex G-75 in 1 M acetic acid yielded a single peak that coeluted with a synthetic E-domain peptide standard. Since secretion of the E-dmnain peptide parallels the expression of IGF-II, the RIA for the prolGF-lI E-domain peptide may he useful for studies of the biosynthesis and secretion of IGF-II under different physiological conditions. The RIA for the IGF-ll E-domain peptide has two technical advantages over the RIA for IGF-II, namely, the lack of interference by IGF binding proteins and the relative ease with which large quantities of pure antigen can he synthesized.
Introduction Insulin-like growth factor II is a single-chain polypeptide mitogenic hormone that is structurally related to proinsulin [1]. The mitogenic activity of this hormone in cell culture suggests that it has a growth-promoting
* Present address: Livingston Research Center, Department of Obstetrics and Gynecology, University of Southern California Medical Center, 1321 N. Mission Road, Los Angeles, CA 90033, U.S.A. Abbreviation: prolGF-II, pro-insulin-like growth factor II. Correspondence: D.S. Straus, Division of Biomedical Sciences, University of California, Riverside, CA 92521-0121, U.S.A.
function in vivo; however, its exact physiological function is not well understood at present. In the rat, the level of expression of the IGF-II gene [2,3] and concentration of IGF-II in plasma [4,5] are high in the fetus and neonate, but decline to very low levels in the adult. High concentrations of IGF-II have also been found in the fetal and neonatal lamb and in the fetal guinea pig [6,7]. These observations suggest that IGF-II may be a major regulator of fetal growth [7,8]. Other evidence suggests an additional role for IGF-II in the central nervous system. IGF-II gene expression in the brain persists into adulthood in rats [2], and IGF-II and variant forms of IGF-II have been found in brain and cerebrospinal fluid in rats and humans [9-11]. These observations suggest a possible growth-promoting or
0167-4889/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
regulatory role for IGF-II in the central nervous system. IGF-II is synthesized as a prohormone precursor that contains an 89 residue carboxyl terminal extension domain (E-domain) that is cleaved off during processing of the hormone [12,13]. The high degree of sequence conservation (79%) between the E-domain regions of rat and human prolGF-II [12,13] has led to speculation that this region may have a biological function. We reported previously the first identification of a naturally occurring peptide fragment of the E-domain region of rat prolGF-II [14]. This peptide was found in media as a secretory product produced by cultured BRL-3A rat liver cells. The E-domain peptide begins at position 117 of the rat prohormone sequence, and its molecular weight determination [15] is consistent with it being equivalent to the carboxyl terminal 40 amino acids of the pro-IGF-II E-domain. We recently synthesized this peptide and developed a radioimmunoassay for the synthetic peptide [15]. This RIA is analogous to the insulin C-peptide RIA, in that it detects a portion of the IGF-II prohormone that is not represented in mature IGF-II. Using this RIA we previously determined that E-domain peptide immunoreactivity was present at high levels in 5-day-old rat pups, but was very low in adult rats [15]. The present study was undertaken to utilize the RIA for further studies of the developmental regulation of the E-domain peptide in rat serum, and to examine the production of this peptide by cultured rat cell lines. Materials and Methods
Animals 3-month-old Sprague-Dawley-derived rats were obtained from Simonsen-Gilroy (Gilroy, CA). The animals were maintained on a 12 h light, 12 h dark cycle and received water and rat chow ad libitum. Rat pups of 5, 11, 15, 20 and 24 days after birth were obtained after breeding in our vivarium. No parental rats were utilized in these studies. All rats were decapitated and serum was immediately stored at - 7 0 ° C until analyzed. Collection of conditioned medium Stock cultures of all cells were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum, 100/~g streptomycin/ml and 70 U penicillin/ml. For collection of conditioned medium, the BRL-3A rat liver cell line [16], REF rat embryo fibroblasts (prepared from rat embryos on day 14 of gestation), N R K normal rat kidney fibroblasts [17], G H 3 rat pituitary tumor cells [18], H4 rat hepatoma cells [19] and HTC rat hepatoma cells [20], were plated at 400 000 cells per 25 cm 2 flask and cultured in MEM supplemented with 100/xg streptomycin/ml and 70 U penicillin/ml. Conditioned medium was collected from flasks
after 2 days of incubation. At that time, cell number was determined by counting with a Coulter counter.
Sephadex G-75 chromatography Conditioned medium (5-10 ml) from BRL-3A or REF cells, serum (2-18 ml) from neonatal or adult rats, or molecular weight standards were acidified with acetic acid to a final concentration of 1 M acetic acid in 20 ml final volume. The sample was centrifuged at 14 000 x g for 15 min and the resulting supernatant (approx. 20 ml) was applied to a Sephadex G-75 column (2.5 x 86 cm for serum; 2.5 x 90 cm for medium) which was equilibrated in 1 M acetic acid. Fractions of 4.3 ml were collected and aliquots of fractions were lyophilized, reconstituted in RIA buffer (0.01 M sodium phosphate, 0.154 M NaC1, 0.02% sodium azide, 0.1% gelatin, 0.1% bovine serum albumin (RIA grade), and 0.01% Triton X-100, pH 7.6), and neutralized prior to measurement of IR-E-peptide. Neutralized aliquots containing precipitates were clarified by centrifugation prior to assay. Peptides Synthetic rat prolGF-IInT_156 (pro-rlGF-II117_156; = E - d o m a i n peptide), and [Tyrn6]pro-rlGF-IllaT_156 (Tyr n6 analog) were synthesized and purified as previously described [15]. Measurement of E-domain peptide binding proteins in serum To examine whether serum contained a binding protein for pro-rlGF-Ii17_156, 20 /~1 of iodinated Tyr n6 analog ((6-8). 105 cpm; approx. 10 ng) was incubated with 1 ml of serum from an adult female rat pool at 4 ° C for 20 h. After the incubation period, the sample was applied to a Sephadex G-75 column (2.5 x 87 cm) previously equilibrated in 0.03 M sodium phosphate, 0.154 M NaC1, 0.02% sodium azide, 0.1% gelatin (pH 7.4). Fractions were collected (5.7 ml) at 4°C, and 3.0 ml aliquots were counted in a gamma counter. In similar experiments, 125I-Tyrn6 analog was incubated with 0.03 M phosphate buffer (as a control), or with an acetic acid/ethanol extract of the adult rat serum pool (see below for extraction methodology) prior to chromatography on Sephadex G-75. For analysis of radioactivity recovered near the void volume, fractions containing the radioactivity were pooled, lyophilized, reconstituted in 20 ml of 1 M acetic acid, and rechromatographed on a Sephadex G-75 column (2.5 x 90 cm) equilibrated with 1 M acetic acid. Competition experiments were performed as described above, with the addition of 10 ~g of unlabeled Tyr 116 analog. Serum extraction Extractions of serum were performed in order to determine the conditions which would eliminate binding
proteins while retaining maximum E-domain peptide. In a preliminary experiment, both iodinated E-domain peptide and an unlabeled E-domain peptide RIA standard were extracted separately with either 12.5% 2 M HC1/87.5% ethanol as per the methodology of Daughaday et al. [21] or with 15% 8 M acetic acid/85% ethanol as described below. After percent recoveries were determined (via % cpm recovered and % recovery of immunoactivity via RIA), it was determined that the acetic acid/ethanol extraction resulted in a higher percentage recovery (76%) of E-domain peptide immunoactivity compared with HC1/ethanol extraction (30%). Thus the acetic acid/ethanol method was used for all extraction experiments. (The % recovery of 125I-labeled E-peptide was higher in the HCl/ethanol method, but the percentage of immunoactivity was much less (30% compared with the the acetic acid/ethanol procedure, indicating loss of E-domain peptide immunoactivity when HC1 was used for extraction.) Acid/ethanol-extracted serum was prepared by mixing 1 ml of adult female rat serum pool with 4 ml of 15% 8 M acetic acid/85% ethanol in polypropylene tubes. Tubes were mixed and centrifuged at 2000 x g for 30 min at 4 ° C. The supernatant was transferred to another polypropylene tube, frozen at - 7 0 ° C and lyophilized. Lyophilized samples were reconstituted in 1 ml of 0.03 M sodium phosphate, 0.154 M NaC1, 0.1% gelatin, 0.02% sodium azide, 0.0025% phenol red and neutralized just prior to binding studies. Lioer extraction Livers were immediately excised and quick-frozen on dry ice. Extraction was performed at 4 ° C using a Dounce homogenizer in 1 M acetic acid. Aliquots (50-100 /~1) of homogenate were lyophilized and later used for protein analysis. Homogenates were centrifuged at 28 000 x g for 1 h at 4 ° C. Supernatants were decanted, frozen at - 7 0 °C and lyophilized. Lyophilized samples were diluted in RIA buffer containing 0.0025% phenol red. Samples were then neutralized, transferred to microfuge tubes, and clarified by centrifugation at top speed in a microfuge for 30 rain at 4 ° C. Supernatants were then assayed for IR-E-domain peptide. In some cases (to demonstrate parallelism of the extracts to the E-domain peptide standard curve) reconstituted samples were diluted prior to RIA. Measurement of protein Protein content of liver homogenates was determined by the Folin phenol method [22] using bovine serum albumin as standard. Lyophilized homogenate was dissolved in hot 0.1 M NaOH prior to analysis. RIA for the E-domain peptide Immunoreactive E-domain peptide was assayed using a polyclonal antibody raised in rabbits as previously
described [15]. The only modification from this procedure was that the incubation was performed at 4 ° C overnight. Bound ligand was separated from free using protein A (Staphylococcus aureus." IgSorb, The Enzyme Center, Boston, MA) to precipitate bound ligand. Statistics Data were analyzed using analysis of variance followed by Duncan's new multiple range test [23]. These procedures were corrected for unequal sample sizes, where appropriate. The significance level was chosen at P < 0.05. Results
The prolGF-II E-domain peptide that we previously identified and for which we developed a radioimmunoassay [14,15] consists of the carboxyl terminal 40 amino acids of the rat prolGF-II E-domain, beginning at position 117 of the prohormone (see Fig. 1). Data in Fig. 2 indicate that serum E-domain peptide levels were very high on day 5 after birth and decreased sharply between days 5 and 25 ultimately to attain very low levels in the adult. The relationship between the immunoreactive material found in neonatal vs. adult serum was analyzed further by comparison of acid-gel chromatographic profiles of immunoreactive material from adult and neonatal sera (Fig. 3a, b). Chromatography of adult rat serum on Sephadex G-75 in 1 M acetic acid yielded a single major broad peak of E-domain peptide immunoactivity that coeluted with a synthetic E-domain peptide standard (Fig. 3a). However, chromatography of day 5 neonatal rat serum yielded 2 peaks of immunoreactivity (Fig. 3b). One of the two peaks of activity (271 ml eluent) coeluted with a synthetic E-domain peptide standard, whereas the second peak (155 ml eluent) eluted in a region where higher molecular weight proteins typically elute (to the left of the cytochrome c standard). Serial dilutions of liver extracts from day 11 and adult rats were shown to displace tracer in parallel to the displacement seen using synthetic E-domain peptide (data not shown). The profile of the concentration of hepatic E-domain peptide from day 11, 15, 20, 24 and adult rats is shown in Fig. 4. Hepatic content of E-domain peptide decreased significantly after day 11, with very low levels measured in the adult liver. Several experiments were performed to determine whether rat serum contained a binding protein for the E-domain peptide. Results from these experiments are shown in Figs. 5a, b and c. In the first series of experiments (Fig. 5a) neutral-gel filtration chromatography was performed with an 125I-E-domain peptide control sample, and this sample was found to elute as a single major peak of radioactivity at 335 ml eluent. However, when the 125I-E-domain peptide was in-
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Fig. 1. The sequence of rat prolGF-lI predicted from cDNA sequence analysis [12,13]. The N-terminal 67 amino acids correspond to the IGF-II hormone. The prohormone also contains a carboxyl terminal extension of 89 residues (68-156) termed the E-domain. The E-domain peptide (which is underlined in the top portion of the figure) represents the carboxyl terminal 40 residues of the E-domain (positions 117-156). Numbers represent residue position.
cubated with 1 ml adult rat serum for 20 h at 4 °C prior to chromatography, four peaks of radioactivity were observed, including two small peaks near the void volume (peaks A and B), a major peak at 335 ml eluent, and a small peak of radioactivity at 545 ml eluent (peak C). Peaks A and B did not diminish even if the incubation of serum and 125I-E-domain peptide was performed in the presence of 10 #g of unlabeled competing peptide (data not shown). This indicates that these two peaks do not represent E-domain peptide complexed with a specific binding protein.
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15
20
25
ADULT
AGE (DAYS AFTER BIRTH)
Fig. 2. E-domain peptide levels in serum of neonatal and adult rats. Adult rats were 4 m o u t h s old. Values represent the m e a n + S.E. of four or five individual samples. F o r adult, each sample = one rat; for neonates, each sample = a pool of four or five pups.
The two small peaks of radioactivity (peaks A and B) that eluted near the void volume were further chromatographed on Sephadex G-75 under acidic conditions (1 M acetic acid), and the profiles of radioactivity in the the resulting fractions are shown in Fig. 5b. The majority of the radioactivity of both peaks remained in the high molecular weight region following acid-gel filtration chromatography indicating that this material did not dissociate in 1 M acetic acid. This provides further evidence that these two peaks do not represent E-domain peptide complexed with a specific binding protein. An additional series of experiments was directed toward determining whether acetic acid/ethanol extraction of rat serum prior to incubation with 125I-E-domain peptide and subsequent neutral-gel filtration chromatography would eliminate peaks A, B, or C that had been generated after incubation with unextracted serum (see Fig. 5a). The results of neutral-gel chromatography of a sample of 125I-E-domain peptide preincubated with acid/ethanol extracted serum are shown in Fig. 5c. Extraction of serum did not eliminate the small peak of radioactivity seen near the void volume (175 ml eluent). However, extraction of serum prior to incubation totally eliminated the peak of radioactivity at 545 ml eluent (peak C, Fig. 5a). Several rat cell lines were examined for their ability to secrete E-domain peptide in culture (Table I). Of the six cell lines examined (BRL-3A rat liver cells, rat
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ELUENT VOLUME (ml) Fig. 3. Chromatographic profiles of adult and neonatal rat serum on Sephadex G-75 in 1 M acetic acid. Absorbance of a 1 : 10 dilution is shown by the dotted line. Solid line, E-domain peptide, normalized to an 18.85 ml sample of serum. The dimensions of the column were 2.5x90 cm. Fractions of 4.3 ml were collected. Molecular weight standards are: cytochrome c (13 320), ribonuclease A (13 680), insulin (5733), bacitracin (1400) and synthetic E-domain peptide (4432). Vt marks the elution position of NaCI. Although not shown in the figure, the ribonuclease A standard ran at 249 ml. (A) An aliquot of pooled adult female rat serum (18.85 mi) was acidified to 1 M acetic acid and chromatographed using 1 M acetic acid as eluent. Aliquots of fractions were lyophilized and reconstituted in RIA buffer prior to assay. The experiment was performed four times (using different serum pools) with identical results. (B) An aliquot (7.5 ml) of pooled day 5 neonatal rat serum (n = 20) was diluted to 18.85 ml and acidified to 1 M acetic acid. Sample was loaded onto the column and eluted with 1 M acetic acid as described above. E-peptide levels have been normalized for an 18.85 ml serum sample for comparison with Fig. 3a. Experiment was performed twice (using two different serum pools) with identical results.
e m b r y o f i b r o b l a s t s ( R E F ) , H 4 a n d H T C h e p a t o m a cells, G H 3 p i t u i t a r y t u m o r cells, a n d a n o r m a l r a t k i d n e y f i b r o b l a s t i c cell l i n e ( N R K ) ) , o n l y B R L - 3 A l i v e r c e l l s and REF fibroblasts produced measurable quantities of E - d o m a i n p e p t i d e . A l t h o u g h B R L - 3 A cells p r o d u c e d 3-5-times more E-domain peptide than REF cells (P < 0.01), b o t h cell t y p e s e x h i b i t e d i n c r e a s e d p r o d u c t i o n o f t h e E - d o m a i n p e p t i d e ( n o r m a l i z e d p e r cell n u m b e r ) when the concentration of fetal bovine serum was inc r e a s e d f r o m 0.3% t o 1.0% ( P < 0.01). To examine and compare the nature of the immunor e a c t i v i t y o f t h e B R L - 3 A vs. R E F cells, s a m p l e s o f m e d i a c o n d i t i o n e d b y t h e s e cells w e r e c h r o m a t o g r a p h e d o n S e p h a d e x G - 7 5 i n 1 M a c e t i c a c i d ( F i g . 6), a n d
profiles were compared. Acid-gel filtration chromatography of conditioned media from BRL-3A or REF cells yielded a single peak of immunoreactivity that coeluted with a synthetic E-domain pep.tide standard (280 ml eluent).
TABLE I Secretion of immunoreactive E-domain peptide from various rat cell lines in culture
Cells were incubated in minimal essential medium for 2 days prior to collection of media, n.d., not detectable; FCS, fetal calf serum. Rat cell line
Origin
% FCS
ng E-Peptide per 105 cells a
BRL-3A
liver
0.3 1.0
72.53+0.52 *'** 145.10 + 7.70 * *
REF
embryo fibroblasts
0.3 1.0
H4
hepatoma
0.3
n.d.
1.0
n.d.
2.0 z o...
~
1.5
GH 3
pituitary tumor
0.3 1.0
n.d. n.d.
NRK
kidney fibroblasts
0.3 1.0
n.d. n.d.
HTC
hepatoma
0.3
n.d.
1.0
n.d.
0,5
0.0
13.34 + 0.10 * 54.80 + 3.91
-
Fig. 4. Analysis of rat liver extracts for content of E-domain peptide. E-domain pcptide levels from extracted livers of neonatal and adult rats. Values represent the mean+S.E, of three or four individual samples. Asterisks indicate significant difference vs. mean values for day 11 values ( P < 0 . 0 5 ) . Mean levels for adult liver were not significantly different compared with levels from livers on day 20.
Values represent the mean+S.E, of three or four independent measurements. * Significantly different ( P < 0 . 0 1 ) from the same cell line in 1% FCS. ** Significantly different ( P < 0.01) from the REF cells at the same percentage of FCS. a
11 m
Discussion
.2 300-
We report here further studies using an RIA for the rat proIGF-II E-domain peptide aimed at determining the nature of the developmental regulation of this peptide in rat serum, and identifying cultured rat cell lines that might secrete this peptide. Previously, we identified this peptide as a naturally occurring secretory product found in rat serum [15] and in medium conditioned by BRL-3A rat liver cells [14,15]. Serum levels of E-domain peptide decreased 60-fold from the day 5 rat pup to the mature adult, and this decline of E-peptide concentrations during the neonatal period is consistent with earlier results on IGF-II in the rat [4,21]. These
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ELUENTVOLUME(ml) Fig. 6. Acid-gel filtration chromatography of m e d i u m conditioned by BRL-3A rat liver cells or by rat embryo fibroblast (REF) cells. Media was diluted to 18.85 ml, acidified to 1 M acetic acid and chromatographed (20 ml) on a 2.5 × 90 cm column of Sephadex G-75 equilibrated with 1 M acetic acid. Fractions of 4.3 ml were collected. Molecular weight markers as in Fig. 3. Aliquots of fractions were lyophilized and assayed for E-peptide content by RIA. Open circles = REF media, right axis; closed circles = BRL-3A media, left axis, Data has been normalized to 18.85 ml conditioned media for the sake of comparison.
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, 700
b O I.*J r-,I.tJ
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data indicate that E-domain peptide levels roughly parallel levels of IGF-II in serum. To investigate further the nature of circulating E-domain peptide during the neonatal period, we subjected serum from day 5 pups and adult rats to gel filtration chromatography in acetic acid. Adult rat serum exhibited a single broad peak of immunoactivity. However, the day 5 neonatal serum profile exhibited two peaks of immunoactivity, one that coeluted with a synthetic E-peptide standard, and a second peak that eluted in a region where higher molecular weight proteins elute (Fig. 3b). We can probably eliminate the idea that this
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200
300 400 500 ELUENT VOLUME (ml)
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700
Fig. 5. Analysis of adult rat serum for E-domain peptide binding proteins. Total recovery of radioactivity from columns ranged from 86-90%. (a) Neutral-gel filtration chromatography on Sephadex G-75 of n~I-E-domain peptide after a 20 h incubation at 4 ° C with: (i) buffer alone (dosed circles) or with 1 ml of normal adult female serum (open circles). The dimensions of the column were 2.5 x 87 cm. Eluent was phosphate buffer (pH 7.4) (see Materials and Methods). Fractions of 5.7 ml were collected. Molecular weight standards were: a-chymotrypsinogen (23240), ribonuclease A (13680) and adenosine triphosphate (551). (b) Acid-gel filtration chromatography of peaks A and B from the experiment shown in a. Peaks from the experiment shown in a were acidified to 1 M acetic acid and re-chromatographed on a 2.5 × 90 cm column of Sephadex G-75 using 1 M acetic acid as eluent. Fractions of 4.3 ml were collected. Open circles represent peak A, and closed circles represent peak B. Molecular weight standards as in Fig. 3. (c) Neutral-gel filtration chromatograpfiy on Sephadex G-75 of nSI-E-domain peptide (closed circles) or 125I-E-domain peptide+ acetic acid/ethanol-extracted adult rat serum (open circles). The dimensions of the column were 2.5 × 87 cm. The column was eluted with phosphate buffer (pH 7.4). Fractions of 5.7 ml were collected. Prior to chromatography, tracer was incubated for 20 h at 4 ° C with either buffer alone or with an acetic a c i d / e t h a n o l extract of 1 ml of adult female rat serum. Molecular weight markers as in a.
12 higher molecular weight material is due to interaction with a binding protein, since the chromatography was performed under acidic conditions (1 M acetic acid). Since the antibody used in the present study is directed against the carboxyl terminus of the proIGF-II E-domain, we suspect that this higher molecular weight material represents the intact proIGF-II molecule. We cannot, however, conclusively rule out the possibility that this material is a large fragment of proIGF-II lacking the amino terminal portion of the proIGF-II molecule. The appearance of the higher molecular weight peak in neonatal serum appears to indicate a difference in processing a n d / o r secretion of proIGF-II in the rat pup as compared with the adult rat. The peak of immunoreactive E-domain peptide obtained with adult rat serum is typically very broad and might conceivably contain a very small amount of high molecular weight material at the extreme leading portion of the peak. However, it is certain that this higher molecular weight material is not present in media conditioned by BRL-3A or REF cells (Fig. 6). The greater relative amount of high molecular weight immunoreactive E-domain material in the neonatal rat as compared with adult rat under acidic chromatographic conditions may indicate: (1) lower activity of IGF-II processing enzymes in rat pup vs. adult rat; (2) over-saturation of the processing enzymes by the large amount of proIGF-II substrate present in rat pup and consequent secretion of the unprocessed hormone; or (3) developmental control over secretion of proIGF-II. In the 5 day neonatal rat, serum E-domain peptide concentrations were very high and declined to low levels in the adult. Hepatic concentrations of E-domain peptide also decreased from high levels in the day 11 neonate to very low levels in the adult rat. The decline in hepatic IGF-II E-domain peptide from neonatal to adult parallels the decline in hepatic IGF-II mRNA observed in other studies [2,3]. We measured immunoreactive E-domain peptide in medium conditioned by various rat cell lines. Although two hepatoma cell lines (H4, HTC) did not secrete measurable E-domain peptide, the BRL-3A liver cells and rat embryo fibroblasts did secrete this peptide. REF cells have also been shown previously to secrete large quantities of IGF-II [24]. Both the BRL-3A and R E F cells were induced to secrete significantly more E-domain peptide on a per cell basis when cultured in medium containing 1% as compared with 0.3% serum. The effect of increased serum on E-domain peptide production was greater with the REFs than with the BRL-3A cells, consistent with the greater serum dependency of the REFs. The stimulatory effect of serum on p r o l G F - I I E-domain peptide production may reflect general metabolic stimulation by serum or may indicate that some component in serum stimulates
E-domain peptide synthesis/secretion from these cells. In regard to the latter possibility, it is of interest to note that Clemmons et al. [25,26] previously observed that platelet-derived growth factor (a component of serum) stimulated the secretion of IGF-I by human fibroblasts. The immunoreactivity of media conditioned by BRL3A or REF cells was shown to be an identical, single chromatographic peak, that coeluted with the synthetic E-domain peptide. The observation that neither intact proIGF-II nor higher molecular weight fragments of the proIGF-II E-domain are detected in conditioned medium using this RIA suggests that proteolytic removal of the carboxyl terminal 40 amino acids of the E-domain (i.e., the E-domain peptide) may be an early step in the processing of proIGF-II in these rat cells. To address the possibility that there might be serum proteins that bind the E-domain peptide, we examined rat serum for evidence of such a protein. Since there is no amino acid sequence homology between IGF-II and the proIGF-II E-domain peptide, it seemed unlikely that IGF-II binding proteins would have affinity for the E-domain peptide. However, it was conceivable that some other protein(s) might bind the E-domain peptide. Overnight incubation of 125I-E-peptide with serum resulted in only a minor shift of radioactivity to the void volume area of the neutral G-75 column (peaks A and B, Fig. 5a). If this shift of activity was due to interaction with a binding protein(s), then acid-gel filtration chromatography should have caused a dissociation of binding protein and tracer, thus resulting in a shift in the elution peak of radioactivity (i.e., 125I-E-domain peptide) back to the position where the synthetic E-domain peptide standard ehited. However, the radioactivity remained near the void volume area after acid-gel chromatography (Fig. 5b), indicating that this minor peak of radioactivity is probably not due to interaction with a binding protein. The radioactivity in the void volume may be an aggregate of tracer, as has been suggested for other peptides [21]. A c i d / e t h a n o l extraction of serum prior to overnight incubation did not eliminate the aggregated material (Fig. 5c, 175 ml eluent). Thus little, if any, binding protein for the E-domain peptide was found in adult rat serum. In contrast, rat serum contains large quantities of IGF-II binding proteins [21]. It was interesting to note that overnight incubation of 125I-E-peptide with serum also generated a radioactive peak that eluted after ATP upon neutral-gel chromatography (peak C, Fig. 5a). That this peak ehited well after ATP (which was used as a low molecular weight standard), suggested that rat serum contained a proteinase that degraded the 12SI-E-domain peptide to a much smaller fragment, and that this degradation product had some affinity for the Sephadex G-75 column. This proteinase was eliminated by extraction of the serum with a c i d / e t h a n o l (compare Fig. 5a with 5c). More experimentation is needed to characterize the
13 n a t u r e of this m i n o r p r o t e i n a s e activity. It is p o s s i b l e that this p r o t e i n a s e activity m a y b e similar to a n acidl a b i l e p r o t e i n a s e activity p r e v i o u s l y d e s c r i b e d in h u m a n s e r u m [27], a l t h o u g h the activity in rat s e r u m a p p e a r s to b e m u c h lower t h a n that p r e v i o u s l y o b s e r v e d in h u m a n serum. Since I G F - I I a n d the E - d o m a i n p e p t i d e are p r o c e s s e d p r o d u c t s o f the s a m e p o l y p e p t i d e p r e c u r s o r molecule, they are synthesized in e q u i m o l a r quantities. O u r s t u d ies to d a t e i n d i c a t e t h a t levels o f the E - d o m a i n p e p t i d e in c u l t u r e m e d i a a n d s e r u m r o u g h l y p a r a l l e l levels of I G F - I I . T h u s the R I A for the E - d o m a i n p e p t i d e will be useful for future studies on the b i o s y n t h e s i s a n d secretion of I G F - I I . This w o u l d be a n a l o g o u s to the R I A for the insulin C - p e p t i d e . R a d i o i m m u n o a s s a y s for the insulin-like g r o w t h factors are p r o b l e m a t i c b e c a u s e of interference b y s e r u m b i n d i n g proteins. This interference is very difficult to e l i m i n a t e c o m p l e t e l y . F o r e x a m p l e , recent studies have d e m o n s t r a t e d that I G F b i n d i n g p r o t e i n s c a n n o t be c o m p l e t e l y r e m o v e d f r o m rat serum [28] o r ovine p l a s m a [29] b y a c i d / e t h a n o l extraction. Thus, the assay for the p r o - I G F - I I E - d o m a i n p e p t i d e has a technical a d v a n t a g e over the R I A for I G F - I I in the lack of i n t e r f e r e n c e b y b i n d i n g proteins. A n a d d i t i o n a l a d v a n t a g e o f the E - d o m a i n p e p t i d e R I A is the relative ease with which large q u a n t i t i e s o f p u r e a n t i g e n can b e synthesized. I n p a r t i c u l a r , the E - d o m a i n p e p t i d e d o e s not c o n t a i n a n y cysteine residues, so that f o l d i n g of the p e p t i d e a n d p r o p e r a l i g n m e n t o f disulfide linkages is n o t a p r o b l e m .
4
5 6 7 8
9 10 11 12 13 14 15 16 17 18 19 20 21
Acknowledgments W e t h a n k N i c h o l e M i h a r a for excellent technical assistance, a n d M a e G o r d o n for her w o r d p r o c e s s i n g skills. This w o r k s u p p o r t e d b y U S D A c o m p e t i t i v e g r a n t 88-37265-4115 a n d N I H g r a n t s R 0 1 - D K 3 9 7 3 9 a n d F32DK-07721.
References 1 Froesch, E.R. and Zapf, J. (1985) Diabetologia 28, 485-493. 2 Brown, A.C., Graham, D.E., Nissley, S.P., Hill, D.J., Strain, A.J. and Rechler, M.M. (1986) J. Biol. Chem. 261, 13144-13150 (and additions: J. Biol. Chem. 262, 2424-2425). 3 Lund, P.K., Moats-Staats, B.M., Hynes, M.A., Simmons, J.G.,
22 23 24 25 26 27 28 29
Jansen, M., D'Ercole, A.J. and Van Wyk, J.J. (1986) J. Biol. Chem. 261, 14539-14544. Moses, A.C., Nissley, S.P., Short, P.A., Rechler, M.M., White, R.M., Knight, A.B. and Higa, O.Z. (1980) Proc. Natl. Acad. Sci. USA 77, 3649-3653. Daughaday, W.H., Parker, K.A., Borowsky, S., Trivedi, B. and Kapadia, M. (1982) Endocrinology 110, 575-581. Gluckman, P.D. and Butler, J.H. (1983) J. Endocrinol. 99, 223-232. Daughaday, W.H., Yanow, C.E. and Kapadia, M. (1986) Endocrinology 199, 490-494. Sara, V.R. and Carlsson-Skwirut, C. (1987) in Highlights on Endocrinology. (Christiansen, C. and Riis, B.J., eds.), pp. 275-280, Bogtrykkeri, Norhaven, A/S. Binoux, M., Hossenlopp, P., Lassarre, C. and Hardouin, N. (1981) FEBS Lett. 124, 178-184. Haselbacher, G. and Humbel, R. (1982) Endocrinology 110, 1822-1824. Haselbacher, G.K., Schwab, M.E., Pasi, A. and Humbel, R.E. (1985) Proc. Natl. Acad. Sci. USA 82, 2153-2157. Dull, T.J., Gray, A., Hayflick, J.S. and Uilrich, A. (1984) Nature (London) 310, 777-781. Whitfield, H.J., Bruni, C.B., Frunzio, R., Terrell, J.E., Nissley, S.P. and Rechler, M.M. (1984) Nature (London) 312, 277-280. Hylka, V.W., Teplow, D.B., Kent, S.B.H. and Straus, D.S. (1985) J. Biol. Chem. 260, 14417-14420. Hylka, V.W., Kent, S.B.H. and Straus, D.S. (1987) Endocrinology 120, 2050-2058. Straus, D.S., Coppock, D.L. and Pang, K.J. (1981) Biochem. Biophys. Res. Commun. 100, 1619-1625. DeLarco, J.E. and Todaro, G.J. (1978) J. Cell Physiol. 94, 335-342. Tashjian, A.H., Jr., Yasumura, Y., Levine, L., Sato, G.H. and Parker, M.L. (1968) Endocrinology 82, 342-352. Straus, D.S. and Takemoto, C. (1987) J. Biol. Chem. 262, 1955-1960. Thompson, E.B., Tomkins, G.M. and Curran, J.F. (1966) Proc. Natl. Acad. Sci. USA 56, 296-303. Daughaday, W.H., Mariz, I.K. and Blethen, S.L. (1980) J. Clin. Endocrinol. Metab. 51,781-788. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Bancroft, T.A. (1968) Topics in Intermediate Statistical Methods. p. 100, Iowa State University Press, Ames, IA. Adams, S.O., Nissley, S.P., Handwerger, S. and Rechler, M.M. (1983) Nature (London) 302, 150-153. Clemmons, D.R., Underwood, L.E. and Van Wyk, J.J. (1981) J. Clin. Invest. 67, 10-19. Clemmons, D.R. (1984) J. Clin. Endocrinol. Metab. 58, 850-856. Powell, D.R., Lee, P.D.K., Chuang, D., Liu, F. and Hintz, R.L. (1987) J. Clin. Endocrinol. Metab. 119, 868-875. Daughaday, W.H., Kapadia, M. and Mariz, I. (1987) J. Lab. Clin. Med. 109, 355-363. Mesiano, S., Young, I.R., Browne, C.A. and Thorburn, G.D. (1988) J. Endocrinol. 119, 453-460.
