Antipeptide antibody to the insulin-like growth factor-I receptor sequence 1232-1246 inhibits the receptor kinase activity.

To approach the question of why insulin-like growth factor-I (IGF-I) and insulin have different physiological actions, we developed antibodies directed against cytoplasmic regions of the IGF-I receptor exhibiting a low degree of homology with the corresponding sequences of the insulin receptor. We found that an antipeptide antibody directed against the beta-subunit carboxyl-terminal sequence (1232-1246) of the IGF-I receptor significantly reduced the in vitro receptor autophosphorylation. The ability of the synthetic peptide corresponding to the IGF-I receptor sequence 1232-1246 to abolish this inhibitory effect reflects the specific nature of the antibody interaction with the targeted domain in the receptor. Antipeptide antibody to IGF-I receptor sequence 1232-1246 also decreased receptor phosphorylation activity toward the exogenous substrate poly(Glu/Tyr). The reduction in poly(Glu/Tyr) phosphorylation was seen even when the antibody was incubated with a receptor previously activated and phosphorylated. Therefore, the inhibitory action on substrate phosphorylation is likely to be unrelated to the antibody reduction of receptor autophosphorylation but rather results from a global decrease in receptor enzymatic activity. The effect of the antipeptide antibody on receptor tyrosine kinase cannot be accounted for by a lowering of the receptor Km for ATP or of its affinity for the substrate poly(Glu/Tyr). Moreover, the interaction of the antibody with the receptor had no repercussion on the ligand binding site as shown by the unaltered IGF-I binding. Taken together our data suggest that the beta-subunit carboxyl-terminal domain of the IGF-I receptor plays a key role in regulating its kinase activity and that the particular sequence recognized by our antipeptide antibody could be involved in negative regulation of receptor functioning.     

Primary structure of rat insulin-like growth factor-I and its biological activities

Rat insulin-like growth factor-I (IGF-I), a serum polypeptide with growth promoting activity, was isolated from rat serum by a combination of acid/ethanol extraction, affinity chromatography, and a series of reversed phase high performance liquid chromatography, cation exchange, and reversed phase. All peptide fragments produced by chymotrypsin digestion of reduced and carboxymethylated rat IGF-I were amino acid sequenced and compared with the sequence of human IGF-I. Three out of 70 of the rat amino acid residues differed from those of human IGF-I as follows: Asp20----Pro, Ser35----Ile and Ala67----Thr. Purified rat IGF-I cross-reacted with polyclonal anti-human IGF-I antibody 75% as compared to human IGF-I, but it cross-reacted only 3% with monoclonal anti-human IGF-I antibody. Thus, it is possible to monitor the metabolic fate of human IGF-I, when injected into rats, without interference by endogenous rat IGF-I. Rat IGF-I showed 65% activity in the radioreceptor, 28.6% activity in the lipogenesis and 22.5% activity in the free fatty acid release inhibition assays as compared to human IGF-I on a protein quantity basis.     

Endogenous IGF-I protects human intestinal smooth muscle cells from apoptosis by regulation of GSK-3 beta activity

We have previously shown that endogenous IGF-I regulates human intestinal smooth muscle cell proliferation by activation of phosphatidylinositol 3 (PI3)-kinase- and Erk1/2-dependent pathways that jointly regulate cell cycle progression and cell division. Whereas insulin-like growth factor-I (IGF-I) stimulates PI3-kinase-dependent activation of Akt, expression of a kinase-inactive Akt did not alter IGF-I-stimulated proliferation. In other cell types, Akt-dependent phosphorylation of glycogen synthase kinase-3 beta (GSK-3 beta) inhibits its activity and its ability to stimulate apoptosis. The aim of the present study was to determine whether endogenous IGF-I regulates Akt-dependent GSK-3 beta phosphorylation and activity and whether it regulates apoptosis in human intestinal muscle cells. IGF-I elicited time- and concentration-dependent GSK-3 beta phosphorylation (inactivation) that was measured by Western blot analysis using a phospho-specific GSK-3beta antibody. Endogenous IGF-I stimulated GSK-3 beta phosphorylation and inhibited GSK-3 beta activity (measured by in vitro kinase assay) in these cells. IGF-I-dependent GSK-3 beta phosphorylation and the resulting GSK-3 beta inactivation were mediated by activation of a PI3-kinase-dependent, phosphoinositide-dependent kinase-1 (PDK-1)-dependent, and Akt-dependent mechanism. Deprivation of serum induced beta-catenin phosphorylation, increased in caspase 3 activity, and induced apoptosis of muscle cells, which was inhibited by either IGF-I or a GSK-3 beta inhibitor. Endogenous IGF-I inhibited beta-catenin phosphorylation, caspase 3 activation, and apoptosis induced by serum deprivation. IGF-I-dependent inhibition of apoptosis, similar to GSK-3 beta activity, was mediated by a PI3-kinase-, PDK-1-, and Akt-dependent mechanism. We conclude that endogenous IGF-I exerts two distinct but complementary effects on intestinal smooth muscle cell growth: it stimulates proliferation and inhibits apoptosis. The growth of intestinal smooth muscle cells is regulated jointly by the net effect of these two processes.     

Effect of Mild Hypoinsulinemia on Renal Hypertrophy: Growth Hormone/Insulin-Like Growth Factor I System in Mild Streptozotocin Diabetes

The metabolic aberrations associated with diabetes mellitus profoundly alter the growth hormone/insulin-like growth factor I (GH/IGF-I) system. In severe experimental diabetes, serum IGF-I level is reduced, reflecting altered hepatic expression. On the other hand, increased levels of kidney IGF-I have been implicated in the development of diabetic kidney disease. This study aimed to examine the effect of mild experimental diabetes with hypoinsulinemia on both the systemic and renal GH/IGF-I systems in a low-dose streptozotocin (STZ)-induced diabetic rat. Diabetic animals with mild hypoinsulinemia developed renal hyperfiltration within 3 days of diabetes, whereas the renal size increased significantly only between 30 and 48 days of diabetes. Plasma GHlevels were unchanged during the entire course of the study, but a decrease in serum IGF-I, IGF-binding protein 3 (IGFBP-3), and IGF-binding protein 4 (IGFBP-4) occurred after 10, 30, and 48 days. Kidney IGF-I and IGF-binding protein 1 (IGFBP-1) mRNA expression increased after 10 and 30 days of diabetes. A significant increase in kidney IGFBP-1/2, IGFBP-3, and IGFBP-4 proteins was seen after 48 days of diabetes.Apositive correlations was found between renal growth and insulin/glucose ratio (r = .57), kidney IGF-I (r = .57), IGFBP-1 mRNA(r = .43), IGFBP-1/2 (r = .41), and IGFBP-4 levels (r = .40). These results demonstrate hyperfiltration within 3 days of diabetes and a similar response in the IGF-I system in mildly and severely hypoinsulinemic rats; however, renomegaly develops slower in mildly diabetic rats at least partly due to delayed changes in the renal IGF and IGF BPs.     

Inhibition of IGFBP-5 binding to extracellular matrix and IGF-I–stimulated DNA synthesis by a peptide fragment of IGFBP-5

Insulin-like growth factor binding protein-5 (IGFBP-5) is synthesized and secreted by smooth muscle cells (SMC). IGFBP-5 synthesis is stimulated five- to sixfold by IGF-I, and IGFBP-5 has been shown to augment IGF-I–stimulated DNA synthesis in this cell type. The ability of IGFBP-5 to augment the SMC response to IGF-I is dependent upon its binding to extracellular matrix. A highly charged region of IGFBP-5 that contains amino acids in positions 201–218 has been shown to mediate binding of IGFBP-5 to human fibroblast extracellular matrix (ECM), and a synthetic peptide containing this sequence inhibits IGFBP-5 binding to fibroblast ECM. In this study we show that exposure of SMC cultures that are constituitively synthesizing IGFBP-5 to a synthetic peptide (termed peptide A) containing this sequence has no effect on its synthesis but reduces its abundance within the ECM. The addition of increasing concentrations of the peptide to SMC cultures resulted in a concentration-dependent reduction in ECM-associated IGFBP-5. In contrast, a control peptide (peptide B), which contained the region of amino acids in positions 131–141 and had a similar charge-to-mass ratio, caused a minimal decrease in ECM binding. This effect was functionally significant since the addition of 10 μg/ ml of peptide A inhibited the cellular replication response to 10 ng/ ml IGF-I by 51%, and peptide B had no effect. The effects of peptide A were not due to nonspecific cytotoxicity since it had no inhibitory effect on the response of these cells to human serum and was associated with only minimal inhibition of the cellular response to platelet-derived growth factor. The findings suggest that inhibiting IGFBP-5 binding to porcine SMC ECM results in reduced cellular responses to IGF-I. J. Cell. Biochem. 71:375–381, 1998. © 1998 Wiley-Liss, Inc.     

Expression of human insulin-like growth factor I in bacteria: use of optimized gene fusion vectors to facilitate protein purification

Several fusions between the gene for human insulin-like growth factor I (IGF-I) and the genes for different IgG-binding fragments of staphylococcal protein A were assembled and compared regarding expression, secretion, and purification of the peptide hormone. After IgG affinity purification of the fusion proteins from the growth medium of Staphylococcus aureus or Escherichia coli, native IGF-I was released by cleavage of an Asn-Gly peptide bond with hydroxylamine. An optimized expression system based on a modified synthetic IgG-binding domain (z), resistant to hydroxylamine, gave the highest yield of fusion protein. After cleavage, the hormone could be separated from the IgG-binding moiety and from noncleaved fusion protein by a second passage through the IgG affinity column. The biological activity and the purity of the IGF-I obtained were confirmed by a radioreceptor assay, N-terminal sequence analysis, polyacrylamide gel electrophoresis, isoelectric focusing, and high-performance liquid chromatography.     

Neutralization of native human gamma interferon (HuIFN gamma) by antibodies to a synthetic peptide encoded by the 5' end of HuIFN gamma cDNA

A synthetic peptide corresponding to the N-terminal amino acid sequence of human gamma-interferon (HuIFN gamma), based on the cDNA sequence, was used to produce antibodies in rabbits that were reactive with native HuIFN gamma. Antibodies from all immunized rabbits neutralized the antiviral activity of HuIFN gamma. Significant neutralization of other HuIFN and mouse IFN was not observed. The peptide had the sequence Cys-Tyr-Cys-Gln-Asp-Pro-Tyr-Val-Lys-Glu-Ala-Glu-Asn-Leu-Lys-Lys-Tyr-Phe-Asn-Ala ,and was coupled to keyhole limpet hemocyanin by disulfide linkage with the use of cystamine. The specificity of the antibodies produced to the peptide was compared to that of antibodies produced to native HuIFN gamma by neutralization of HuIFN gamma and by reactivity with peptide in the enzyme-linked immunosorbent assay (ELISA). The ratio of anti-peptide antibody neutralization of HuIFN gamma vs reactivity with peptide in the ELISA was at least 28-fold lower than for anti-HuIFN gamma antibody. Thus the antibodies to peptide and to HuIFN gamma were directed primarily against different determinants on native HuIFN gamma or the anti-HuIFN gamma antiserum probably contained antibodies to additional determinants. The anti-peptide antibodies should be useful for further characterization and purification of HuIFN gamma.     

Chronic effect of insulin-like growth factor I on renin synthesis, secretion, and renal function in fetal sheep

In the adult, insulin-like growth factor I (IGF-I) increases glomerular filtration rate (GFR) and renal blood flow (RBF) during both acute and chronic treatment. To study its effects on the developing kidney, chronically catheterized fetal sheep (120 +/- 1 days gestation) were infused intravenously for up to 10 days with 80 microgram/h IGF-I (n = 5) or vehicle (0.1% BSA in saline, n = 6). In contrast to previous acute studies in adult rats and humans, after 4 h of IGF-I fetal GFR and RBF were unchanged. Fractional sodium reabsorption increased (P < 0.05). However, by 4 days, GFR per kilogram had risen by 35 +/- 13% (P < 0.05), whereas RBF remained unchanged. Tubular growth and maturation may have occurred, as proximal tubular sodium reabsorption increased by ~35% (P < 0.005). Therefore, despite a marked increase in filtered sodium (~30%, P < 0.05), fractional sodium reabsorption did not change. Although the effects of IGF-I on renal function were delayed, plasma renin activity and concentration were both elevated after 4 h and remained high at 4 days (P < 0.05). Despite this, arterial pressure and heart rate did not change. Kidneys of IGF-I-infused fetuses weighed ~30% more (P = 0.05) and contained ~75% more renin than control fetuses (P < 0.005). Thus, in the fetus, the renal effects of long-term IGF-I infusion are very different from the adult, possibly because IGF-I stimulated kidney growth.     

Expressed protein ligation using an N-terminal cysteine containing fragment generated in vivo from a pelB fusion protein

Advances in expressed protein ligation (EPL) methods that permit specific introduction of unique modifications into proteins have facilitated protein engineering, structure-function and protein interaction studies. An EPL-generated hybrid exchangeable apolipoprotein has been constructed from recombinant fragments of apolipoprotein E (apoE) and apolipophorin III (apoLp-III). A recombinant fusion protein comprised of human apoE N-terminal residues 1-111, a modified Saccharomyces cerevisiae intein and a chitin binding domain was subjected to 2-mercaptoethanesulfonic acid (MESNA) induced cleavage to generate apoE(1-111)-MESNA. A second fusion protein was comprised of a bacterial pelB leader peptide fused to a variant form of Galleria mellonella apoLp-III residues 1-91. The N-terminal pelB leader sequence directed the newly synthesized fusion protein to the Escherichia coli perisplamic space where endogenous leader peptidase cleavage generated the desired N-terminal cysteine-containing protein fragment. The resulting apoLp-III fragment, which contained no sequence tags or tails, escaped the bacteria and accumulated in the culture medium. When cultured in M9 minimal medium, Asp1Cys apoLp-III(1-91) was produced in high yield and was the sole major protein in the culture supernatant. Ligation reactions with apoE(1-111)-MESNA yielded an engineered hybrid apolipoprotein. The results document the utility of the pelB fusion protein system for generating active N-terminal cysteine containing proteins for EPL applications.     

An antifungal peptide from Phaseolus vulgaris cv. brown kidney bean

A 5.4-kDa antifungal peptide, with an N-terminal sequence highly homologous to defensins and inhibitory activity against Mycosphaerella arachidicola (IC(50)= 3 μM), Setospaeria turcica and Bipolaris maydis, was isolated from the seeds of Phaseolus vulgaris cv. brown kidney bean. The peptide was purified by employing a protocol that entailed adsorption on Affi-gel blue gel and Mono S and finally gel filtration on Superdex 75. The antifungal activity of the peptide against M. arachidicola was stable in the pH range 3-12 and in the temperature range 0°C to 80°C. There was a slight reduction of the antifungal activity at pH 2 and 13, and the activity was indiscernible at pH 0, 1, and 14. The activity at 90°C and 100°C was slightly diminished. Deposition of Congo red at the hyphal tips of M. arachidicola was induced by the peptide indicating inhibition of hyphal growth. The lack of antiproliferative activity of brown kidney bean antifungal peptide toward tumor cells, in contrast to the presence of such activity of other antifungal peptides, indicates that different domains are responsible for the antifungal and antiproliferative activities.     

Stimulation of macrophage growth and multinucleated cell formation in rat bone marrow cultures by insulin-like growth factor I

In this study the effects of rhIGF-I on macrophage differentiation and growth have been studied using liquid suspension cultures of rat bone marrow cells. IGF-I stimulated macrophage growth in a dose-dependent manner, a maximum response was found at a concentration of 20 ng/ml. IGF-I effects could be ascribed to stimulation of both postmitotic and proliferating cells. A remarkable finding was that IGF-I induced formation of multinucleated cells (MNC). The MNC resembled macrophage-like cells (AcP, NSE positive). A monoclonal antibody to rhIGF-I significantly inhibited IGF-stimulated macrophage growth and MNC formation. A specific antibody to mouse CSF-1 reduced IGF-stimulated macrophage growth in mouse bone marrow cultures indicating that IGF-I effects could, at least in part, be ascribed to endogenous production of CSF-1. These findings indicate that IGF-I in concert with locally induced CSF-1 can influence the differentiation and growth of bone marrow-derived macrophages.     

Defining the Antigenic Diversity of Plasmodium falciparum Apical Membrane Antigen 1 and the Requirements for a Multi-Allele Vaccine against Malaria

Apical Membrane Antigen 1 (AMA1) is a leading malaria vaccine candidate and a target of naturally-acquired human immunity. Plasmodium falciparum AMA1 is polymorphic and in vaccine trials it induces strain-specific protection. This antigenic diversity is a major roadblock to development of AMA1 as a malaria vaccine and understanding how to overcome it is essential. To assess how AMA1 antigenic diversity limits cross-strain growth inhibition, we assembled a panel of 18 different P. falciparum isolates which are broadly representative of global AMA1 sequence diversity. Antibodies raised against four well studied AMA1 alleles (W2Mef, 3D7, HB3 and FVO) were tested for growth inhibition of the 18 different P. falciparum isolates in growth inhibition assays (GIA). All antibodies demonstrated substantial cross-inhibitory activity against different isolates and a mixture of the four different AMA1 antibodies inhibited all 18 isolates tested, suggesting significant antigenic overlap between AMA1 alleles and limited antigenic diversity of AMA1. Cross-strain inhibition by antibodies was only moderately and inconsistently correlated with the level of sequence diversity between AMA1 alleles, suggesting that sequence differences are not a strong predictor of antigenic differences or the cross-inhibitory activity of anti-allele antibodies. The importance of the highly polymorphic C1-L region for inhibitory antibodies and potential vaccine escape was assessed by generating novel transgenic P. falciparum lines for testing in GIA. While the polymorphic C1-L epitope was identified as a significant target of some growth-inhibitory antibodies, these antibodies only constituted a minor proportion of the total inhibitory antibody repertoire, suggesting that the antigenic diversity of inhibitory epitopes is limited. Our findings support the concept that a multi-allele AMA1 vaccine would give broad coverage against the diversity of AMA1 alleles and establish new tools to define polymorphisms important for vaccine escape.     

Evidence for Production of a New Lantibiotic (Butyrivibriocin OR79A) by the Ruminal Anaerobe Butyrivibrio fibrisolvens OR79: Characterization of the Structural Gene Encoding Butyrivibriocin OR79A

The ruminal anaerobe Butyrivibrio fibrisolvens OR79 produces a bacteriocin-like activity demonstrating a very broad spectrum of activity. An inhibitor was isolated from spent culture fluid by a combination of ammonium sulfate and acidic precipitations, reverse-phase chromatography, and high-resolution gel filtration. N-terminal analysis of the isolated inhibitor yielded a 15-amino-acid sequence (G-N/Q-G/P-V-I-L-X-I-X-H-E-X-S-M-N). Two different amino acid residues were detected in the second and third positions from the N terminus, indicating the presence of two distinct peptides. A gene with significant homology to one combination of the determined N-terminal sequence was cloned, and expression of the gene was confirmed by Northern blotting. The gene (bvi79A) encoded a prepeptide of 47 amino acids and a mature peptide, butyrivibriocin OR79A, of 25 amino acids. Significant sequence homology was found between this peptide and previously reported lantibiotics containing the double-glycine leader peptidase processing site. Immediately downstream of bvi79A was a second, partial open reading frame encoding a peptide with significant homology to proteins which are believed to be involved in the synthesis of lanthionine residues. These findings indicate that the isolated inhibitory peptides represent new lantibiotics. Results from both total and N-terminal amino acid sequencing indicated that the second peptide was identical to butyrivibriocin OR79A except for amino acid substitutions in positions 2 and 3 of the mature lantibiotic. Only a single coding region was detected when restriction enzyme digests of total DNA were probed either with an oligonucleotide based on the 5′ region of bvi79A or with degenerate oligonucleotides based on the predicted sequence of the second peptide.     

Epitope mapping by amino-acid-sequence-specific antibodies reveals that both ends of the alpha subunit of Na+/K(+)-ATPase are located on the cytoplasmic side of the membrane.

Right-side-out vesicles of pig kidney microsomes and amino-acid-sequence-specific antibodies were used to probe the sidedness of the C-terminus and the N-terminus of the catalytic alpha subunit of Na+/K(+)-ATPase. Polyclonal antibodies were raised in rabbits against the peptide corresponding to the N-terminal sequence GRDKYEPAAVSE (peptide 1-12) and against peptides corresponding to the C-terminal sequences IFVYDEVRKLIIRRR (peptide 991-1005) and RPGGWVEKETYY (peptide 1005-1016). These antibodies were purified by affinity chromatography on the respective peptide-Sepharose columns. Moreover, antibodies against the N-terminal dodecapeptide GRDKYEPAAVSE were obtained by affinity purification from heteroclonal antibodies against the alpha subunit of pork kidney Na+/K(+)-ATPase. These antibodies reacted with native as well as SDS-denaturated Na+/K(+)-ATPase. When the antibodies were used to probe the sidedness of the sequences in right-side-out vesicles of pig kidney microsomes, the N-terminal peptide 1-12 as well as the C-terminal peptides 991-1005 and 1005-1016 were found on the cytosolic side. Concanavalin A, however, which interacts with the beta subunit, a glycoprotein, reacted with the outside of right-side-out vesicles.     

Monoclonal antibodies distinguish synthetic peptides that differ in one chemical group

By using human calcitonin (hCT), human calcitonin-gene-related peptide (hCGRP), and a synthetic peptide with a sequence analogous to the 34 C-terminal amino acids of human preprocalcitonin (designated as PQN-34) as haptens in the generation of monoclonal antibodies, we assessed the role of amido and amino groups in paratope-epitope binding. By using peptide inhibition experiments and solid-phase immunoassays, monoclonal anti-hCT antibody CT07 and monoclonal anti-hCGRP antibody CGR01 were found to bind to an antigenic determinant located in the C-terminal segment of the hormones. These epitopes comprise the seven C-terminal amino acids of the hormones, and the presence of the hormone-ending carboxamide group was found to be essential for antibody binding. The corresponding heptapeptides, either bearing a carboxyl group or else linked to a glycine residue at their C-terminal part, failed to react with the antibodies. Moreover, these monoclonal antibodies did not bind to synthetic peptides analogous to the C-terminal region of the hormone precursor molecules that comprised the epitope site flanked by a peptide sequence. In an attempt to assess whether amido groups when present on the side-chain of amino acids may also modulate antibody binding, a monoclonal antibody referred to as QPO1 was produced and was found to recognize an antigenic determinant localized in the N-terminal region of the PQN-34 peptide bearing a glutamine residue as the N-terminal amino acid. The epitope was found to correspond to a topographic assembled site, and binding of QPO1 was found to be substantially dependent on the presence of the free amino and the side-chain amido groups borne by the N-terminal glutamine residue of this peptide PQN-34. In contrast to these findings, an antigenic determinant located in the internal sequence of calcitonin and recognized by monoclonal anti-hCT antibody CT08 was found to be expressed on the mature form of the hormone, as well as on synthetic peptides with sequence mimicking that of preprocalcitonin. These data should guide the choice of synthetic peptide haptens for the production of anti-protein antibodies.     

Antibodies to deduced sequences of the insulin receptor distinguish conserved and nonconserved regions in the IGF-I receptor

Antipeptide antibodies directed to two amino acid sequences predicted from the cDNA encoding the insulin proreceptor have been used to study the relationship between the human receptors for insulin and insulin-like growth factor I (IGF-I). An antibody directed to a cytoplasmic domain near the membrane spanning region of the proreceptor inhibited the protein tyrosine kinase activity of both receptors whereas an antibody directed to the C terminus of the insulin receptor showed no cross-reactivity with the IGF-I receptor. The results establish that the cloned cDNA from the human placenta encodes the insulin receptor and not the closely related IGF-I receptor, that the IGF-I and insulin receptors share a specific amino acid sequence necessary for the expression of enzymatic activity, and that the C terminus of the insulin receptor is not conserved in the IGF-I receptor.     

Increases in free, unbound insulin-like growth factor I enhance insulin responsiveness in human hepatoma G2 cells in culture

Insulin-like growth factor-binding protein (IGFBP)-1 binds to insulin-like growth factor (IGF)-I and -II with high affinity and has been shown to modulate IGF-I actions in vivo and in vitro. The synthesis of IGFBP-1 is suppressed by insulin, and administration of IGFBP-1 to rats results in impaired glucose metabolism. A synthetic peptide (bp1-01) has been shown to have a high affinity and specificity for human IGFBP-1 and to inhibit IGF-I binding. The current studies were undertaken to determine if, after incubation of bp1-01 with IGF-I.IGFBP-1 complexes, anabolic and insulin-like effects of IGF-I could be detected in human hepatoma (HepG2) cell cultures and to determine the receptor subtype(s) through which these effects were mediated. Incubation of HepG2 cells with bp1-01 (200 nm) increased IGF-I-stimulated protein synthesis by 44% and glycogen synthesis by 170% compared with stimulation by IGF-I alone. Incubation with bp1-01 also enhanced IGF-I-stimulated tyrosine phosphorylation of the IGF-I/insulin hybrid receptor and insulin receptor substrate 1. Exposure of the cells to bp1-01 alone enhanced glycogen synthesis and phosphorylation of IGF-I/insulin hybrid receptors. This was not a direct effect of bp1-01 because it did not bind to the receptor and did not activate tyrosine kinase activity in the presence of an anti-IGF-I receptor antibody. The addition of bp1-01 (200 nm) plus insulin to HepG2 cell culture medium resulted in increased tyrosine phosphorylation of the hybrid receptor, insulin receptor substrate 1, and the glycogen synthesis response compared with the effects of insulin alone. This enhancement of hybrid receptor phosphorylation and glycogen synthesis by bp1-01 peptide was diminished by preincubation with an inhibitory antibody for the alpha subunit of IGF-I receptor (alphaIR3). bp1-01 stimulated the hybrid receptor phosphorylation response to IGF-I, and this effect was inhibited by prior incubation of the cells with alphaIR3. In conclusion, bp1-01 competes with IGF-I for binding to IGFBP-1, which leads to release of free IGF-I from IGF-I.IGFBP-1 complexes. This released IGF-I stimulates biologic actions that are mediated predominantly through the IGF-I/insulin hybrid receptor.     

Fast protein sequencing of monoclonal antibody by real-time digestion on emitter during nanoelectrospray

Growth in the pharmaceutical industry has led to an increasing demand for rapid characterization of therapeutic monoclonal antibodies. The current methods for antibody sequence confirmation (e.g., N-terminal Edman sequencing and traditional peptide mapping methods) are not sufficient; thus, we developed a fast method for sequencing recombinant monoclonal antibodies using a novel digestion-on-emitter technology. Using this method, a monoclonal antibody can be denatured, reduced, digested, and sequenced in less than an hour. High throughput and satisfactory protein sequence coverage were achieved by using a non-specific protease from Aspergillus saitoi, protease XIII, to digest the denatured and reduced monoclonal antibody on an electrospray emitter, while electrospray high voltage was applied to the digestion mixture through the emitter. Tandem mass spectrometry data was acquired over the course of enzyme digestion, generating similar information compared to standard peptide mapping experiments in much less time. We demonstrated that this fast protein sequencing method provided sufficient sequence information for bovine serum albumin and two commercially available monoclonal antibodies, mouse IgG1 MOPC21 and humanized IgG1 NISTmAb. For two monoclonal antibodies, we obtained sequence coverage of 90.5–95.1% for the heavy chains and 98.6–99.1% for the light chains. We found that on-emitter digestion by protease XIII generated peptides of various lengths during the digestion process, which was critical for achieving sufficient sequence coverage. Moreover, we discovered that the enzyme-to-substrate ratio was an important parameter that affects protein sequence coverage. Due to its highly automatable and efficient design, our method offers a major advantage over N-terminal Edman sequencing and traditional peptide mapping methods in the identification of protein sequence, and is capable of meeting an ever-increasing demand for monoclonal antibody sequence confirmation in the biopharmaceutical industry.     

Compensatory glomerular growth after unilateral nephrectomy is VEGF dependent

Various growth factors and cytokines have been implicated in different forms of kidney enlargement. Vascular endothelial growth factor (VEGF) is essential for normal renal development and plays a role in diabetic glomerular enlargement. To explore a possible role for VEGF in compensatory renal changes after uninephrectomy, we examined the effect of a neutralizing VEGF-antibody (VEGF-Ab) on glomerular volume and kidney weight in mice treated for 7 days. Serum and kidney insulin-like growth factor I (IGF-I) levels were measured, since IGF-I has been implicated in the pathogenesis of compensatory renal growth, and VEGF has been suggested to be a downstream mediator of IGF-I. Placebo-treated uninephrectomized mice displayed an early transient increase in kidney IGF-I concentration and an increase in glomerular volume and kidney weight. In VEGF-Ab-treated uninephrectomized animals, increased glomerular volume was abolished, whereas renal hypertrophy was partially blocked. Furthermore, the renal effects of VEGF-Ab administration were seen without affecting the renal IGF-I levels. In conclusion, these results demonstrate that compensatory glomerular growth after uninephrectomy is VEGF dependent.     

Characterization of insulin-like growth factor I receptors on Madin-Darby canine kidney (MDCK) cell line

Specific insulin-like growth factor I (IGF-I) receptors on the Madin-Darby canine kidney (MDCK) cell line were identified and characterized. [125I]IGF-I specifically bound to the cells, but [125I]insulin bindings to the cells was minimal. Unlabeled IGF-I displaced both the IGF-I and insulin bindings with potencies that were 100 and 10 times as great as insulin. By an affinity labeling technique, IGF type I receptors were present in the MDCK cells. IGF-I stimulated DNA synthesis and cell proliferation at physiological concentrations. On the other hand, insulin had a little effect on DNA synthesis. These data suggest that IGF type I receptors as demonstrated in MDCK cells are involved in DNA synthesis and cell proliferation.