Biochemical determinants of the IGFBP-3-hyaluronan interaction.

IGFBP-3, the most abundant IGFBP and the main carrier of insulin-like growth factor I (IGF-I) in the circulation, can bind IGF-1 with high affinity, which attenuates IGF/IGF-IR interactions, thereby resulting in antiproliferative effects. The C-terminal domain of insulin-like growth factor-binding protein-3 (IGFBP-3) is known to contain an 18-basic amino acid motif capable of interacting with either humanin (HN) or hyaluronan (HA). We previously showed that the 18-amino acid IGFBP-3 peptide is capable of binding either HA or HN with comparable affinities to the full-length IGFBP-3 protein and that IGFBP-3 can compete with the HA receptor, CD44, for binding HA. Blocking the interaction between HA and CD44 reduced viability of A549 human lung cancer cells. In this study, we set out to better characterize IGFBP-3-HA interactions. We show that both stereochemistry and amino acid identity are important determinants of the interaction between the IGFBP-3 peptide and HA and for the peptide's ability to exert its cytotoxic effects. Binding of IGFBP-3 to either HA or HN was unaffected by glycosylation or reduction of IGFBP-3, suggesting that the basic 18-amino acid residue sequence of IGFBP-3 remains accessible for interaction with either HN or HA upon glycosylation or reduction of the full-length protein. Removing N-linked oligosaccharides from CD44 increased its ability to compete with IGFBP-3 for binding HA, while reduction of CD44 rendered the protein relatively ineffective at blocking IGFBP-3-HA interactions. We conclude that both deglycosylation and disulfide bond formation are important for CD44 to compete with IGFBP-3 for binding HA.

IGFBP-3/IGFBP-3 Receptor System as an Anti-Tumor and Anti-Metastatic Signaling in Cancer.

Insulin-like growth factor binding protein-3 (IGFBP-3) is a p53 tumor suppressor-regulated protein and a major carrier for IGFs in circulation. Among six high-affinity IGFBPs, which are IGFBP-1 through 6, IGFBP-3 is the most extensively investigated IGFBP species with respect to its IGF/IGF-I receptor (IGF-IR)-independent biological actions beyond its endocrine/paracrine/autocrine role in modulating IGF action in cancer. Disruption of IGFBP-3 at transcriptional and post-translational levels has been implicated in the pathophysiology of many different types of cancer including breast, prostate, and lung cancer. Over the past two decades, a wealth of evidence has revealed both tumor suppressing and tumor promoting effects of IGF/IGF-IR-independent actions of IGFBP-3 depending upon cell types, post-translational modifications, and assay methods. However, IGFBP-3's anti-tumor function has been well accepted due to identification of functional IGFBP-3-interacting proteins, putative receptors, or crosstalk with other signaling cascades. This review mainly focuses on transmembrane protein 219 (TMEM219), which represents a novel IGFBP-3 receptor mediating antitumor effect of IGFBP-3. Furthermore, this review delineates the potential underlying mechanisms involved and the subsequent biological significance, emphasizing the clinical significance of the IGFBP-3/TMEM219 axis in assessing both the diagnosis and the prognosis of cancer as well as the therapeutic potential of TMEM219 agonists for cancer treatment.

Insulin-like growth factor binding protein 3 gene of golden pompano (TroIGFBP3) promotes antimicrobial immune defense.

Insulin-like growth factor binding protein 3 (IGFBP3), an important member of the IGFBP family, plays an important biological role in regulating cellular proliferation, differentiation, growth, apoptosis, and innate immunity. However, studies concerning IGFBP3 in teleosts are very limited and IGFBP3 function remains unclear. In this study, we conducted both in vivo and in vitro functional analyses of an IGFBP3 (TroIGFBP3) from the teleost fish golden pompano (Trachinotus ovatus). TroIGFBP3 is composed of 286 amino acid residues and shares a high amino acid sequence similarity (50.18%-93.71%) with other IGFBP3 sequences in humans and teleosts. TroIGFBP3 was widely distributed in various tissues, with the highest expression in the liver. TroIGFBP3 expression was significantly upregulated following Vibrio harveyi infection. The results of in vitro assays showed that TroIGFBP3 could stimulate macrophage activation and promote peripheral blood leukocytes (PBLs) proliferation. Meanwhile, TroIGFBP3 overexpression significantly inhibited bacterial infection in fish tissues, whereas TroIGFBP3 knockdown resulted in increased bacterial dissemination and colonization in golden pompano tissues in vivo. Furthermore, recombinant TroIGFBP3 could inhibit cellular proliferation and promote apoptosis of mouse tumor cells. Taken together, these results indicated that TroIGFBP3 plays a significant role in innate antibacterial immunity and provides a theoretical foundation for investigating the function of IGFBP3 in fish immune response.

Exosomes derived from miR-375-overexpressing human adipose mesenchymal stem cells promote bone regeneration.

OBJECTIVES: The present study aimed to investigate whether exosomes derived from miR-375-overexpressing human adipose mesenchymal stem cells (hASCs) could enhance bone regeneration. MATERIALS AND METHODS: Exosomes enriched with miR-375 (Exo [miR-375]) were generated from hASCs stably overexpressing miR-375 after lentiviral transfection and identified with transmission electron microscopy, nanosight and western blotting. The construction efficiency of Exo (miR-375) was evaluated with qRT-PCR and incubated with human bone marrow mesenchymal stem cells (hBMSCs) to optimize the effective dosage. Then, the osteogenic capability of Exo (miR-375) was investigated with ALP and ARS assays. Furthermore, dual-luciferase reporter assay and western blotting were conducted to reveal the underlying mechanism of miR-375 in osteogenic regulation. Finally, Exo (miR-375) were embedded with hydrogel and applied to a rat model of calvarial defect, and µ-CT analysis and histological examination were conducted to evaluate the therapeutic effects of Exo (miR-375) in bone regeneration. RESULTS: miR-375 could be enriched in exosomes by overexpressing in the parent cells. Administration of Exo (miR-375) at 50 µg/mL improved the osteogenic differentiation of hBMSCs. With miR-375 absorbed by hBMSCs, insulin-like growth factor binding protein 3 (IGFBP3) was inhibited by binding to its 3'UTR, and recombinant IGFBP3 protein reduced the osteogenic effects triggered by Exo (miR-375). After incorporated with hydrogel, Exo (miR-375) displayed a slow and controlled release, and further in vivo analysis demonstrated that Exo (miR-375) enhanced the bone regenerative capacity in a rat model of calvarial defect. CONCLUSIONS: Taken together, our study demonstrated that exosomes derived from miR-375-overexpressing hASCs promoted bone regeneration.

Higher Maternal Protein Intake during Pregnancy Is Associated with Lower Cord Blood Concentrations of Insulin-like Growth Factor (IGF)-II, IGF Binding Protein 3, and Insulin, but Not IGF-I, in a Cohort of Women with High Protein Intake.

Background: Prenatal exposure to dietary protein may program growth-regulating hormones, consequently influencing early-life growth patterns and later risk of associated chronic diseases. The insulin-like growth factor (IGF) axis is of particular interest in this context given its influence on pre- and postnatal growth and its sensitivity to the early nutritional environment.Objective: Our objective was to examine associations of maternal protein intake during pregnancy with cord blood concentrations of IGF-I, IGF-II, IGF binding protein-3 (IGFBP-3), and insulin.Methods: We studied 938 mother-child pairs from early pregnancy through delivery in the Project Viva cohort. Using multivariable linear regression models adjusted for maternal race/ethnicity, education, income, smoking, parity, height, and gestational weight gain and for child sex, we examined associations of second-trimester maternal protein intake [grams per kilogram (weight before pregnancy) per day], as reported on a food frequency questionnaire, with IGF-I, IGF-II, IGFBP-3, and insulin concentrations in cord blood. We also examined how these associations may differ by child sex and parity.Results: Mothers were predominantly white (71%), college-educated (64%), and nonsmokers (67%). Mean ± SD protein intake was 1.35 ± 0.35 g ⋅ kg-1 ⋅ d-1 Each 1-SD increment in second-trimester protein intake corresponded to a change of -0.50 ng/mL (95% CI: -2.26, 1.26 ng/mL) in IGF-I and -0.91 µU/mL (95% CI: -1.45, -0.37 µU/mL) in insulin. Child sex and parity modified associations of maternal protein intake with IGF-II and IGFBP-3: protein intake was inversely associated with IGF-II in girls (P-interaction = 0.04) and multiparous mothers (P-interaction = 0.05), and with IGFBP-3 in multiparous mothers (P-interaction = 0.04).Conclusions: In a cohort of pregnant women with relatively high mean protein intakes, higher intake was associated with lower concentrations of growth-promoting hormones in cord blood, suggesting a pathway that may link higher protein intake to lower fetal growth. This trial was registered at clinicaltrials.gov as NCT02820402.

Antagonists of IGF:Vitronectin Interactions Inhibit IGF-I-Induced Breast Cancer Cell Functions.

We provide proof-of-concept evidence for a new class of therapeutics that target growth factor:extracellular matrix (GF:ECM) interactions for the management of breast cancer. Insulin-like growth factor-I (IGF-I) forms multiprotein complexes with IGF-binding proteins (IGFBP) and the ECM protein vitronectin (VN), and stimulates the survival, migration and invasion of breast cancer cells. For the first time we provide physical evidence for IGFBP-3:VN interactions in breast cancer patient tissues; these interactions were predominantly localized to tumor cell clusters and in stroma surrounding tumor cells. We show that disruption of IGF-I:IGFBP:VN complexes with L(27)-IGF-II inhibits IGF-I:IGFBP:VN-stimulated breast cancer cell migration and proliferation in two- and three-dimensional assay systems. Peptide arrays screened to identify regions critical for the IGFBP-3/-5:VN and IGF-II:VN interactions demonstrated IGFBP-3/-5 and IGF-II binds VN through the hemopexin-2 domain, and VN binds IGFBP-3 at residues not involved in the binding of IGF-I to IGFBP-3. IGFBP-interacting VN peptides identified from these peptide arrays disrupted the IGF-I:IGFBP:VN complex, impeded the growth of primary tumor-like spheroids and, more importantly, inhibited the invasion of metastatic breast cancer cells in 3D assay systems. These studies provide first-in-field evidence for the utility of small peptides in antagonizing GF:ECM-mediated biologic functions and present data demonstrating the potential of these peptide antagonists as novel therapeutics. Mol Cancer Ther; 15(7); 1602-13. ©2016 AACR.

Humanin Peptide Binds to Insulin-Like Growth Factor-Binding Protein 3 (IGFBP3) and Regulates Its Interaction with Importin-ß.

Nuclear translocation of IGFBP3 by importin-ß1 is a prerequisite for IGFBP3-induced apoptosis. The neuroprotective peptide humanin (HN) counteracts IGFBP3-induced cell death. However, the mechanism by which humanin protects cells is currently unknown. The natural synthesis of this peptide decreases with age, coincident with the likelihood for the development of Alzheimer's Disease, making it a promising target for therapeutics. We have examined the effect of full-length humanin and a synthetic analogue (HN 3-19), known to be sufficient for its neuroprotective function, on the interaction between IGFBP3 and importin-ß1. Using competitive ligand dot blotting, co-immunoprecipitation, and an ELISA-based binding assay, we determined that 1) humanin binds to IGFBP3 with a Kd of 5.05 µM and 2) both humanin (IC50 of 18.1 µM) and HN 3-19 (IC50 of 10.3 µM) interfere with the binding of importin-ß1 to IGFBP3 in vitro. We also demonstrated that HN 3-19 is able to reduce the rate of apoptosis in a human lung adenocarcinoma cell line, suggesting a possible mechanism of action for humanin as an inhibitor of IGFBP3 nuclear translocation. Understanding the exact mechanism by which humanin and its analogue, HN 3-19, bind to IGFPB3 and regulate its interaction with importin-ß1 will open the door to modulating the protein-protein interactions involved in neuronal cell death.

Insulin-Like Growth Factor Binding Proteins--an Update.

The insulin-like growth factor (IGF) system is essential for normal growth and development, and its perturbation is implicated in a number of diseases. IGF activity is finely regulated by a family of six high-affinity IGF binding proteins (IGFBPs). 1GFBPs usually inhibit IGF actions but may enhance them under certain conditions. Additionally, IGFBPs bind non-IGF ligands in the extracellular space, cell membrane, cytoplasm and nucleus, thereby modulating cell proliferation, survival and migration in an IGF-independent manner. IGFBP activity is regulated by transcriptional mechanisms as well as by post-translational modifications and proteolysis. Understanding the balance between the various actions of IGFBPs in vivo may lead to novel insights into disease processes and possible IGFBP-based therapeutics.

Amyloid beta-mediated epigenetic alteration of insulin-like growth factor binding protein 3 controls cell survival in Alzheimer's disease.

Swedish double mutation (KM670/671NL) of amyloid precursor protein (APP) is reported to increase toxic amyloid ß (Aß) production via aberrant cleavage at the ß-secretase site and thereby cause early-onset Alzheimer's disease (AD). However, the underlying molecular mechanisms leading to AD pathogenesis remains largely unknown. Previously, our transcriptome sequence analyses revealed global expressional modifications of over 600 genes in APP-Swedish mutant-expressing H4 (H4-sw) cells compared to wild type H4 cells. Insulin-like growth factor binding protein 3 (IGFBP3) is one gene that showed significantly decreased mRNA expression in H4-sw cells. In this study, we investigated the functional role of IGFBP3 in AD pathogenesis and elucidated the mechanisms regulating its expression. We observed decreased IGFBP3 expression in the H4-sw cell line as well as the hippocampus of AD model transgenic mice. Treatment with exogenous IGFBP3 protein inhibited Aß1-42- induced cell death and caspase-3 activity, whereas siRNA-mediated suppression of IGFBP3 expression induced cell death and caspase-3 cleavage. In primary hippocampal neurons, administration of IGFBP3 protein blocked apoptotic cell death due to Aß1-42 toxicity. These data implicate a protective role for IGFBP3 against Aß1-42-mediated apoptosis. Next, we investigated the regulatory mechanisms of IGFBP3 expression in AD pathogenesis. We observed abnormal IGFBP3 hypermethylation within the promoter CpG island in H4-sw cells. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine restored IGFBP3 expression at both the mRNA and protein levels. Chronic exposure to Aß1-42 induced IGFBP3 hypermethylation at CpGs, particularly at loci -164 and -173, and subsequently suppressed IGFBP3 expression. Therefore, we demonstrate that expression of anti-apoptotic IGFBP3 is regulated by epigenetic DNA methylation, suggesting a mechanism that contributes to AD pathogenesis.

Quantitative glycoproteomics analysis reveals changes in N-glycosylation level associated with pancreatic ductal adenocarcinoma.

Glycosylation plays an important role in epithelial cancers, including pancreatic ductal adenocarcinoma. However, little is known about the glycoproteome of the human pancreas or its alterations associated with pancreatic tumorigenesis. Using quantitative glycoproteomics approach, we investigated protein N-glycosylation in pancreatic tumor tissue in comparison with normal pancreas and chronic pancreatitis tissue. The study lead to the discovery of a roster of glycoproteins with aberrant N-glycosylation level associated with pancreatic cancer, including mucin-5AC (MUC5AC), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), insulin-like growth factor binding protein (IGFBP3), and galectin-3-binding protein (LGALS3BP). Pathway analysis of cancer-associated aberrant glycoproteins revealed an emerging phenomenon that increased activity of N-glycosylation was implicated in several pancreatic cancer pathways, including TGF-ß, TNF, NF-kappa-B, and TFEB-related lysosomal changes. In addition, the study provided evidence that specific N-glycosylation sites within certain individual proteins can have significantly altered glycosylation occupancy in pancreatic cancer, reflecting the complexity of the molecular mechanisms underlying cancer-associated glycosylation events.

Quantification of binding of IGF-1 to BI 836845, a candidate therapeutic antibody against IGF-1 and IGF-2, and effects of this antibody on IGF-1:IGFBP-3 complexes in vitro and in male C57BL/6 mice.

IGF-1 and IGF-2 are potent mitogens acting through the IGF-1 receptor (IGF-1R). The importance of the IGF system in neoplasia has been demonstrated in several models, and IGF-1 signaling has become a target for drug development. The drug candidate BI 836845 is a fully human IgG1 ligand-neutralizing antibody that cross-reacts with IGF-1 and IGF-2. It has been shown to reduce both IGF-1R phosphorylation and cellular proliferation in preclinical studies. In rodent studies, administration of BI 836845 leads to large increases in total IGF-1 concentration in serum, despite reduced serum IGF-1 activity as measured by a kinase activation assay. Despite the fact that anti-IGF-ligand antibodies have entered clinical trials, their effect on IGF-binding proteins has not been described. In this report, we developed a novel technique to measure ligand-BI 836845 binding, and we apply it to a mouse model in various contexts. We show that although large increases in total serum IGF-1 levels are observed, the vast majority of ligand is present as a complex with BI 836845, and total serum IGF-binding protein-3 levels are decreased. Finally, we show that BI 836845 treatment induces an increase in GH levels, a finding consistent with attempted compensation at the level of the pituitary. Our results reveal complexities in the physiologic sequelae of BI 836845 administration that have implications for determination of optimal dosing regimens and for development of pharmacodynamic endpoints for clinical trials.

Core functional sequence of C-terminal GAG-binding domain directs cellular uptake of IGFBP-3-derived peptides.

The current study clarifies the role of the Glycosaminoglycan (GAG)-binding domain of insulin-like growth factor binding protein-3 (IGFBP-3) in cell penetration. The cell penetration function of IGFBP-3 has been mapped to an 18-residue GAG-binding domain in the C-terminal region that mobilizes cellular uptake and nuclear localization of unrelated proteins. Uptake of KW-22, a 22-residue peptide that encompasses the 18-residue GAG-binding domain, and another IGFBP-3 peptide carrying a streptavidin protein cargo was investigated in Chinese hamster ovary (CHO) cells defective at several steps of biosynthesis of cell surface GAGs. The severity of GAG truncation was highly correlated to the impairment of uptake ranging from complete abrogation to only a partial reduction, suggesting that GAG-binding is required for uptake. The 18-residue GAG-binding domain consists of an 8-residue KK-8 basic sequence devoid of Arg and an adjacent 10-residue QR-10 sequence rich in Arg. Peptide mapping of uptake and GAG-binding activities within the KW-22 peptide showed that the 8-residue KK-8 basic peptide retained 80% of GAG-binding activity with no uptake activity while the 10-residue QR-10 peptide retained 53% of uptake activity and 18% of GAG-binding activity. This suggests that KK-8 carries out the majority of GAG-binding function while QR-10 carries out the majority of the cell entry function. To our knowledge, this is the first report of physical separation of the uptake and GAG-binding functions within a short cell penetrating peptide and may shed light on the general mechanism of uptake of Arg-rich CPPs and guide new design of Arg-rich CPP-assisted drug/gene delivery systems.

IGF dependent modulation of IGF binding protein (IGFBP) proteolysis by pregnancy-associated plasma protein-A (PAPP-A): multiple PAPP-A-IGFBP interaction sites.

BACKGROUND: Pregnancy-associated plasma protein-A (PAPP-A) is a local regulator of insulin-like growth factor (IGF) bioavailability in physiological systems, but many structural and functional aspects of the metzincin metalloproteinase remain to be elucidated. PAPP-A cleaves IGF binding protein (IGFBP)-4 and IGFBP-5. Cleavage of IGFBP-4, but not IGFBP-5, depends on the binding of IGF before proteolysis by PAPP-A can occur. The paralogue PAPP-A2 has two substrates among the six IGFBPs: IGFBP-3 and IGFBP-5. METHODS: Sets of chimeric proteins between IGFBP-4 and -5, and IGFBP-3 and -5 were constructed to investigate the structural requirements for IGF modulation. At the proteinase level, we investigated the importance of individual acidic amino acids positioned in the proteolytic domain of PAPP-A for proteolytic activity against IGFBP-4 and -5. Interaction between PAPP-A and its substrates was analyzed by surface plasmon resonance. RESULTS AND CONCLUSION: We provide data suggesting that the C-terminal domain of the IGFBPs is responsible for IGF-dependent modulation of access to the scissile bond. Loss or reduction of IGFBP proteolysis by PAPP-A was observed upon mutation of residues positioned in the unique 63-residue stretch separating the zinc and Met-turn motifs, and in the short sequence following the Met-turn methionine. A model of the proteolytic domain of PAPP-A suggests the presence of structural calcium ions in the C-terminal subdomain, implicated in IGFBP substrate interactions. GENERAL SIGNIFICANCE: Detailed knowledge of interactions between PAPP-A and its substrates is required to understand the modulatory role of PAPP-A on IGF receptor stimulation.

The importance of metal ions for the formation and isolation of insulin-like growth factor-binding protein 3-transferrin (IGFBP-3-Tf) complexes, and the analysis of their physiological involvement.

Insulin-like growth factor (IGF) binding protein 3 (IGFBP-3) is the most abundant IGFBP in the circulation and its main role is to regulate the amount of free, receptor-reactive IGFs that are involved in a broad range of metabolic and mitogenic activities. IGFBP-3 interacts with several other biomolecules forming complexes that might have IGF-dependent or independent functions. Transferrin (Tf) forms complexes with IGFBP-3, but the physiological significance of this interaction remains unclear. Tf is an iron-transporting protein, and is able to carry other metal ions as well. IGFBP-3, on the other hand, possesses one metal-binding domain. As both proteins have the ability to bind metals, the formation of the IGFBP-3-Tf complexes may be expected to influence the metabolism of metal ions and/or mitogenic/metabolic roles in which IGFBP-3 is involved. The aim of this study was to quantify the amount of complexes in the circulation, to investigate the importance of a specific metal ion for their formation and isolation, and to search for the possible physiological place of the IGFBP-3-Tf complexes. The effects of the following ions were analyzed: Fe(3+), Cu(2+), Ni(2+), Mg(2+) and Zn(2+). Results have shown that the amount of IGFBP-3 involved in the complex formation with Tf in healthy persons was 5.4 ± 1.02 nM and iron ions were substantial for their formation and isolation without employment of harsh conditions. The amount of IGFBP-3-Tf complexes was further determined in persons with iron over-load, patients with iron-deficiency anemia and patients with colorectal carcinoma-associated anemia. The amount of complexes was in direct correlation with the concentrations of iron and IGFBP-3. In the case of the colorectal carcinoma-associated anemia, the amount of complexes was inversely correlated with the concentration of ferritin. IGFBP-3, thus, appears to be a member of a network of iron-binding proteins that participates in cell signaling that involves iron-associated response.

Posttranslational modifications of the insulin-like growth factor-binding protein 3 in patients with type 2 diabetes mellitus assessed by affinity chromatography.

Structural and ligand-binding properties of the insulin-like growth factor-binding protein (IGFBP)-3 in patients with poorly controlled diabetes mellitus type 2 were investigated using boronic acid- and lectin-affinity chromatography. IGFBP-3 species separated by chromatography were analyzed by immunoblotting and surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS). Increased IGFBP-3 binding to boronic acid in patients was shown to be accompanied by the increased ligand-binding. Increased binding of IGFBP-3 forms to lectins from Sambucus nigra (SNA) and Canavalia ensiformis (ConA) in patients, on the other hand, was either not accompanied by altered ligand-binding (in the case of ConA) or it was reduced (in the case of SNA). Strong and opposite effects of glycation and additional sialylation on ligand binding qualify them as factors that may be involved in the regulation of the amount of free, physiologically active IGFs, and modulation of processes that accompany development and progression of diabetes. SELDI-TOF MS analysis revealed a fragment of 13.9 kDa as representative for the non-glycosylated form of IGFBP-3, whereas a fragment of 28.0 kDa profiled as typical for the glycosylated/glycated IGFBP-3 species. The same fragmentation pattern found in healthy persons and in patients indicates that the same degradation process predominantly occurs in both groups of individuals.

Colocalization and identification of interaction sites between IGFBP-3 and GalNAc-T14.

GalNAc-T14 was identified as a novel IGFBP-3 binding partner in previous studies. Here, we furtherly confirmed the interaction between them by confocal microscopy, and identified the binding domain and probable interaction sites of GalNAc-T14 with IGFBP-3. The result of subcellular localization indicated that GalNAc-T14 was distributed in the cytosol, whereas IGFBP-3 existed in the cytosol and nucleolus. Confocal analyses demonstrated that IGFBP-3 and GalNAc-T14 colocalized in the cytosol. The result from yeast two hybrid assay showed that the C terminus of GalNAc-T14 (408-552aa) was essential for the interaction between GalNAc-T14 and IGFBP-3, especially Tyr(408), Pro(409), and Glu(410) of GalNAc-T14 may play key roles in the interaction with IGFBP-3. In conclusion, these studies demonstrated that IGFBP-3 and GalNAc-T14 are colocalized in MCF-7 cells and confirmed the interaction between IGFBP-3 and GalNAc-T14. This interaction may play an important role in the functional regulation of IGFBP-3.

Circulating salmon 41-kDa insulin-like growth factor binding protein (IGFBP) is not IGFBP-3 but an IGFBP-2 subtype.

In vertebrates, most circulating insulin-like growth factor (IGF) is bound to multiple forms of IGF-binding proteins (IGFBPs) that differ both structurally and functionally. In mammals, the largest reservoir of IGF in the circulation comes from a large (150kDa) ternary complex comprised of IGF bound to IGFBP-3, which is bound to an acid label subunit (ALS), and this variant of IGFBP is regulated by growth hormone (GH) and feed intake. Although multiple variants of IGFBPs ranging from 20 to 50kDa have been found in fishes, no ternary complex is present and it has been assumed that the majority of circulating IGF is bound to fish IGFBP-3. Consistent with this assumption is previous work in salmon showing the presence of a 41-kDa IGFBP that is stimulated by GH, decreases with fasting and increases with feeding. However, the hypothesis that the salmon 41-kDa IGFBP is structurally homologous to mammalian IGFBP-3 has not been directly tested. To address this issue, we cloned cDNAs for several Chinook salmon IGFBPs, and found that the cDNA sequence of the 41-kDa IGFBP is most similar to that of mammalian IGFBP-2 and dissimilar to IGFBP-3. We found an additional IGFBP (termed IGFBP-2a) with high homology to mammalian IGFBP-2. These results demonstrate that salmon 41-kDa IGFBP is not IGFBP-3, but a paralog of IGFBP-2 (termed IGFBP-2b). Salmon IGFBP-2s are also unique in terms of having potential N-glycosylation sites and splice variants. Additional research on non-mammalian IGFBPs is needed to fully understand the molecular/functional evolution of the IGFBP family and the significance of the ternary complex in vertebrates.

Alterations of insulin-like growth factor binding protein 3 (IGFBP-3) glycosylation in patients with breast tumours.

OBJECTIVES: Insulin-like growth factor binding protein-3 (IGFBP-3) is an important modulator of development and progression of breast cancer as it regulates the amount of free, physiologically active IGF-I and IGF-II. Changes in the glycosylation pattern within IGFBP-3 may affect its interaction with ligands. The aim of this study was to investigate whether such changes occur during disease progression. DESIGN AND METHODS: IGFBP-3 in serum samples from healthy women and from women with breast tumours was characterised in terms of its concentration (IRMA), glycosylation moiety (lectin-affinity chromatography) and distribution of molecular species (immunoblotting). RESULTS: In patients with benign tumours the concentration and carbohydrate content of IGFBP-3 was unaltered compared to healthy women. In patients with malignant tumours in most cases these two parameters were unchanged, but there were women whose concentration of IGFBP-3 was reduced and its structure was altered. In non-surviving cancer patients the concentration of IGFBP-3 was significantly reduced and these molecules contained a greater amount of biantennary complex type N-glycans having more mannose, fucose, bisecting GlcNAc and terminal sialic acid residues. CONCLUSION: Our results showed that breast cancer progression causes alterations of IGFBP-3 glycosylation. The extent of changes increases with breast cancer severity.

Identification of IGFBP-3 fragments generated by KLK2 and prevention of fragmentation by KLK2-inhibiting peptides.

Kallikrein-related peptidase 2 (KLK2) degrades insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) in vitro. IGFBP-3 forms complexes with IGFs, preventing them from binding to their receptors and stimulating cell proliferation and survival. IGF-independent actions have also been described for IGFBP-3. The degradation of IGFBP-3 by KLK2 or other proteases in the prostate may promote the growth of prostate cancer. We studied IGFBP-3 degradation by immunoblotting and two specific immunoassays, one recognizing only native non-fragmented IGFBP-3 and the other one recognizing both intact and proteolytically cleaved IGFBP-3. Peptides were used to inhibit the enzyme activity of KLK2 and cleavage sites in IGFBP-3 were identified by mass spectrometry. KLK2 proteolyzed IGFBP-3 into several small fragments, mostly after Arg residues, in keeping with the trypsin-like activity of KLK2. The fragmentation could be inhibited by KLK2-inhibiting peptides in a dose-dependent fashion. As degradation of IGFBP-3 could lead to a more aggressive cancer phenotype, inhibition of KLK2 activity might be useful for treatment of prostate cancer and other diseases associated with increased KLK2 activity.

Activity of human pregnancy insulin-like growth factor binding protein-3: determination by reconstituting recombinant complexes.

During pregnancy, IGF binding protein-3 (IGFBP-3) is completely proteolyzed to fragments with low affinities for IGFs but appears to transport IGFs normally in high-molecular-mass complexes. We previously reported that synthetic isolated amino- and carboxyl-terminal domains of IGFBP-3 cooperate to bind IGFs, and we investigated whether this is the mechanism whereby proteolyzed IGFBP-3 fragments bind IGFs normally in pregnancy serum. Two fragments of IGFBP-3 have been isolated from pregnancy serum, one with the same N-terminal sequence as intact IGFBP-3 (GASSG) and the other with an N-terminal sequence (160)KVDYE. Recombinant forms of these proteins, IGFBP-3(1-159) and IGFBP-3(160-264), have been synthesized and characterized, demonstrating that although the fragments individually have greatly reduced affinity for IGF complex formation, when combined they cooperate to form complexes with IGF with or without the acid-labile subunit, inhibit IGF transport across endothelial cell monolayers and inhibit IGF-I-induced IGF type I receptor phosphorylation. It is proposed that proteolysis of IGFBP-3 into two discrete complementary fragments does not significantly increase IGF bioavailability, consistent with previous findings that proteolyzed IGFBP-3 in pregnancy serum is functionally normal and circulates as part of the IGF ternary complexes.