The Effects of 20-kDa Human Placental GH in Male and Female GH-deficient Mice: An Improved Human GH?

A rare 20K isoform of GH-V (here abbreviated as GHv) was discovered in 1998. To date, only 1 research article has characterized this isoform in vivo, observing that GHv treatment in male high-fat fed rats had several GH-like activities, but unlike GH lacked diabetogenic and lactogenic activities and failed to increase IGF-1 or body length. Therefore, the current study was conducted to further characterize the in vivo activities of GHv in a separate species and in a GH-deficient model (GH-/- mice) and with both sexes represented. GHv-treated GH-/- mice had significant increases to serum IGF-1, femur length, body length, body weight, and lean body mass and reduced body fat mass similar to mice receiving GH treatment. GH treatment increased circulating insulin levels and impaired insulin sensitivity; in contrast, both measures were unchanged in GHv-treated mice. Since GHv lacks prolactin receptor (PRLR) binding activity, we tested the ability of GH and GHv to stimulate the proliferation of human cancer cell lines and found that GHv has a decreased proliferative response in cancers with high PRLR. Our findings demonstrate that GHv can stimulate insulin-like growth factor-1 and subsequent longitudinal body growth in GH-deficient mice similar to GH, but unlike GH, GHv promoted growth without inhibiting insulin action and without promoting the growth of PRLR-positive cancers in vitro. Thus, GHv may represent improvements to current GH therapies especially for individuals at risk for metabolic syndrome or PRLR-positive cancers.

Receptor expression by JEG-3 trophoblast cells in the presence of placenta secreted factors.

Preeclampsia still remains one of the most severe pregnancy complications and is an actual problem in the obstetrics practice. At present, the joint impact of cytokines and other placenta secreted factors on trophoblast cell functional activity during preeclampsia complicated pregnancy remains unclear. The aim of the study is to estimate the surface receptors expression by trophoblast cells in the presence of placenta secreted factors during physiological pregnancy and at preeclampsia. Trophoblast cells of the JEG-3 line were incubated in the presence of supernatants obtained by cultivation of placentas from women with physiological pregnancy and with preeclampsia. Surface receptors expression by trophoblast cells was estimated by FACS Canto II flow cytometer. It was established that in the third trimester both under normal and pathological conditions, the placenta secreted factors impact on the cytokine receptor expression by trophoblast differs while the trophoblast response capacity to the migration and proliferation stimulating and inhibiting signals remains stable. JEG-3 line cells enhanced the expression of CD186, CD140a, Integrin ß6, VE-cadherin, CD29, and CD140a in the case of incubation in the presence of placenta supernatants from the third-trimester pregnancy complicated with preeclampsia compared to incubation in the presence of placenta supernatants form the third trimester of physiological pregnancy.

The Placental Variant of Human Growth Hormone Reduces Maternal Insulin Sensitivity in a Dose-Dependent Manner in C57BL/6J Mice.

The human placental GH variant (GH-V) is secreted continuously from the syncytiotrophoblast layer of the placenta during pregnancy and is thought to play a key role in the maternal adaptation to pregnancy. Maternal GH-V concentrations are closely related to fetal growth in humans. GH-V has also been proposed as a potential candidate to mediate insulin resistance observed later in pregnancy. To determine the effect of maternal GH-V administration on maternal and fetal growth and metabolic outcomes during pregnancy, we examined the dose-response relationship for GH-V administration in a mouse model of normal pregnancy. Pregnant C57BL/6J mice were randomized to receive vehicle or GH-V (0.25, 1, 2, or 5 mg/kg · d) by osmotic pump from gestational days 12.5 to 18.5. Fetal linear growth was slightly reduced in the 5 mg/kg dose compared with vehicle and the 0.25 mg/kg groups, respectively, whereas placental weight was not affected. GH-V treatment did not affect maternal body weights or food intake. However, treatment with 5 mg/kg · d significantly increased maternal fasting plasma insulin concentrations with impaired insulin sensitivity observed at day 18.5 as assessed by homeostasis model assessment. At 5 mg/kg · d, there was also an increase in maternal hepatic GH receptor/binding protein (Ghr/Ghbp) and IGF binding protein 3 (Igfbp3) mRNA levels, but GH-V did not alter maternal plasma IGF-1 concentrations or hepatic Igf-1 mRNA expression. Our findings suggest that at higher doses, GH-V treatment can cause hyperinsulinemia and is a likely mediator of the insulin resistance associated with late pregnancy.

Review: Endocrine regulation of placental phenotype.

Hormones have an important role in regulating fetal development. They act as environmental signals and integrate tissue growth and differentiation with relation to nutrient availability. While hormones control the developmental fate of resources available to the fetus, the actual supply of nutrients and oxygen to the fetus depends on the placenta. However, much less is known about the role of hormones in regulating placental development, even though the placenta has a wide range of hormone receptors and produces hormones itself from early in gestation. The placenta is, therefore, exposed to hormones by autocrine, paracrine and endocrine mechanisms throughout its lifespan. It is known to adapt its phenotype in response to environmental cues and fetal demand signals, particularly when there is a disparity between the fetal genetic drive for growth and the nutrient supply. These adaptive responses help to maintain fetal growth during adverse conditions and are likely to depend, at least in part, on the hormonal milieu. This review examines the endocrine regulation of placental phenotype with particular emphasis on the glucocorticoid hormones. It focuses on the availability of placental hormone receptors and on the effects of hormones on the morphology, transport capacity and endocrine function of the placenta.

Induction of hepatic CYP3A enzymes by pregnancy-related hormones: studies in human hepatocytes and hepatic cell lines.

CYP3A activity is induced by approximately 2-fold during the third trimester of human pregnancy. Placental growth hormone (PGH), estrogens (primarily 17ß-estradiol), cortisol, and progesterone have the potential to modulate CYP3A activity. Therefore, we determined whether the elevated plasma concentrations of these hormones during pregnancy induce hepatic CYP3A expression. We incubated sandwich-cultured human hepatocytes (SCHH) from premenopausal female donors (n = 2) with the physiologic (unbound, 1× total) and the 10× total third trimester hormone plasma concentrations (individually and in combination) and determined their effect on CYP3A activity and the transcripts of CYP3A4, CYP3A5, and the respective hormone receptors (growth hormone receptor, glucocorticoid receptor, and estrogen receptor alpha). Of all the hormones, cortisol was the most potent inducer of CYP3A activity and CYP3A4, CYP3A5 mRNA expression. The combination of PGH/growth hormone and cortisol induced CYP3A activity and expression significantly more than did cortisol alone. When incubated with the unbound or total plasma concentration of all the hormones, CYP3A activity in SCHH was induced to an extent comparable to that observed in vivo during the third trimester. These hormones had only a modest effect on the mRNA expression of the hormone receptors. The pattern of induction observed in SCHH was reproduced in HepaRG cells but not in HuH7/HepG2 cells. SCHH or HepaRG cells could be used to determine the mechanistic basis of CYP3A induction during pregnancy and to predict the magnitude of induction likely to be observed during the first and second trimesters, when phenotyping studies to measure in vivo CYP3A activity are logistically difficult to perform.

Site-specific PEGylation endows a mammalian ribonuclease with antitumor activity.

Mammalian ribonucleases are emerging as cancer chemotherapeutic agents. Their cationicity engenders cell permeability, and their enzymatic activity destroys the biochemical information encoded by RNA. The pharmacologic potential of ribonucleases is, however, obviated by their high sensitivity to a cytosolic inhibitor protein (RI) and their small size, which limits their residence in serum. We reasoned that site specific conjugation of a poly(ethylene glycol) (PEG) chain could both reduce sensitivity to RI and increase serum half-life. We found that appending a PEG moiety can enable bovine pancreatic ribonuclease (RNase A) to evade RI, depending on the site of conjugation and the length and branching of the chain. Although a pendant PEG moiety decreases antiproliferative activity in vitro, PEGylation discourages renal clearance in vivo and leads to nearly complete tumor growth inhibition in a mouse xenograft model. These data demonstrate that a pendant PEG moiety can be beneficial to the action of proteins that act within the cytosol, and that strategic site-specific PEGylation can endow a mammalian ribonuclease with potent antitumor activity.

20-kDa placental hGH-V has diminished diabetogenic and lactogenic activities compared with 22-kDa hGH-N while retaining antilipogenic activity.

Placental human growth hormone-variant (hGH-V) and pituitary human growth hormone-N (hGH-N) are of identical size (22 kDa) but differ in 13 residues scattered throughout the protein. Several isoforms of GH are produced by the hGH-N and hGH-V genes including a 20-kDa hGH-V resulting from a 45-bp deletion caused by the use of an alternative acceptor site within exon 3. To date, the biological properties of the 20-kDa GH-V have not been characterized in vivo. Using young male Wistar rats fed either chow or a high-fat (HF) diet for 4 wk postweaning, we investigated the effect of 7 days treatment with either 22-kDa hGH-N, 20-kDa hGH-V (5 ug x g(-1) x day(-1) sc), or vehicle on body composition and endocrine and metabolic profiles. Total body growth (absolute weight gain and linear growth trajectory) in the 20-kDa hGH-V-treated animals was intermediary between that of control and hGH-N-treated animals. Both 22-kDa hGH-N and 20-kDa hGH-V significantly reduced total body fat mass compared with control animals, and there were no differences between the GH isoforms in anti-lipogenic activity in animals fed the HF diet. Fasting plasma insulin and C peptide were significantly increased in animals on the HF diet and further increased by hGH-N but were unchanged in 20-kDa hGH-V-treated animals compared with saline-treated controls. Plasma volume as assessed by hematocrit was increased in hGH-N-treated animals but was unchanged in 20-kDa hGH-V-treated animals compared with controls. Furthermore, 20-kDa hGH-V had reduced lactogenic (prolactin receptor mediated) activity characteristic of hGH-N as tested in vitro compared with the 20-kDa hGH-N and 22-kDa hGH-N variants. In summary, placental 20-kDa hGH-V retains some of the growth-promoting and all antilipogenic activities of pituitary 22-kDa hGH-N but has diminished diabetogenic and lactogenic properties compared with the native 22-kDa hGH-N.

Large-scale preparation and in vitro characterization of biologically active human placental (20 and 22K) and pituitary (20K) growth hormones: placental growth hormones have no lactogenic activity in humans.

Expression plasmids containing DNA sequences optimized for expression in Escherichia coli were prepared encoding human pituitary (hGH-N 20K) and placental (hGH-V 20 and 22K) growth hormones. The proteins were expressed in bacteria, refolded and purified to homogeneity by anion-exchange chromatography on Q-Sepharose according to a unique protocol developed for each protein. The yields from 5l of fermentation culture varied between 400 and 700mg of electrophoretically pure, over 95% monomeric protein. Circular dichroism (CD) analysis revealed similarity of the purified hGHs' secondary structure to that of the pituitary hGH-N 22K, except for hGH-V 20K, in which the alpha-helix content was lower. The purified proteins were stable as a 0.1% sterile solution held at pH 10-11 at 4 degrees C for at least one month. All three purified hGH molecules formed a 1:2 complex with hGH receptor extracellular domain (hGHR-ECD), similar to hGH-N 22K. Binding experiments using hGHR-ECD revealed that the differences between the two 22K variants or between the two 20K variants were not significant, except that hGH-V 20K exhibited slightly lower affinity. Somatogenic activity was tested in vitro using FDC-P1 cell lines. Whereas the bioactivity of 22K hGHs and hGH-N 20K in FDC-P1-9D11 cells stably transfected with hGHR was almost equal and two to threefold higher than that of hGH-V 20K, in FDC-P1 3B9 cells stably transfected with rabbit (rb) GHR, the bioactivity of both 20K analogues was significantly (five to ninefold) lower than that of the 22K hormones. The lactogenic activity measured in heterologous assays (Nb2-11C cells and Baf/3 cells stably transfected with the long form of rabbit prolactin receptor) revealed that the activity of hGH-N 20K was close to that of hGH-N 22K in the Baf/3 cells, but 4.5-fold lower in the Nb2 cells. The activity of hGH-V 22K was ninefold less in Nb2 cells and 55-fold less in Baf/3 cells, whereas hGH-V 20K had no lactogenic activity in either bioassay. In contrast, in a homologous lactogenic assay using Baf/3 LP cells stably transfected with hPRLR, the activity of both placental hGHs was nil and the activity of hGH-N 20K was 4.3-fold lower than that of hGH-N 22K. The latter finding raises the question of whether the lack of intrinsic lactogenic activity in the placental hGHs that dominate during pregnancy has any physiological relevance.

Radical scavenging activity of ribonuclease inhibitor from cow placenta.

Cow placenta ribonuclease inhibitor (CPRI) has been purified 5062-fold by affinity chromatography, the product being homogeneous by sodium dodecyl sulfate-gel electrophoresis. The chemiluminescence technique was used to determine the radical scavenging activities of CPRI toward different reactive oxygen species (ROS) including superoxide anion (O2-*), hydroxyl radical (OH*), lipid-derived radicals (R*), and singlet oxygen (1O2). CPRI could effectively scavenge O2-*, OH*, R*, and 1O2 at EC50 of 0.12, 0.008, 0.009, and 0.006 mg/ml, respectively. In addition, the radical scavenging activities of CPRI were higher than those of tea polyphenols, indicating that CPRI is a powerful antioxidant.

Cytotoxicity of bovine seminal ribonuclease: monomer versus dimer.

Bovine seminal ribonuclease (BS-RNase) is a homologue of bovine pancreatic ribonuclease (RNase A). Unlike RNase A, BS-RNase has notable toxicity for human tumor cells. Wild-type BS-RNase is a homodimer linked by two intermolecular disulfide bonds. This quaternary structure endows BS-RNase with resistance to inhibition by the cytosolic ribonuclease inhibitor protein (RI), which binds tightly to RNase A and monomeric BS-RNase. Here, we report on the creation and analysis of monomeric variants of BS-RNase that evade RI but retain full enzymatic activity. The cytotoxic activity of these monomeric variants exceeds that of the wild-type dimer by up to 30-fold, indicating that the dimeric structure of BS-RNase is not required for cytotoxicity. Dimers of these monomeric variants are more cytotoxic than wild-type BS-RNase, suggesting that the cytotoxicity of the wild-type enzyme is limited by RI inhibition following dissociation of the dimer in the reducing environment of the cytosol. Finally, the cytotoxic activity of these dimers is less than that of the constituent monomers, indicating that their quaternary structure is a liability. These data provide new insight into structure-function relationships of BS-RNase. Moreover, BS-RNase monomers described herein are more toxic to human tumor cells than is any known variant or homologue of RNase A including Onconase, an amphibian homologue in phase III clinical trials for the treatment of unresectable malignant mesothelioma.

Production of recombinant human placental variant growth hormone in Pichia pastoris.

cDNA encoding mature human placental variant growth hormone (HGH-V) was synthesized by retro-transcription polymerase chain reaction (RT-PCR) from total RNA recovered from human term-placenta and cloned in pBluescript plasmid (pBS) in Escherichia coli. cDNA was subcloned into pPIC9, fusing it to the flanking regulatory sequences of the Pichia pastoris alcohol oxidase 1 gene (AOX1) and finally introduced into the genome of this yeast by homologous recombination. The resulting new recombinant strain produced and secreted, towards the culture medium, mature HGH-V, whose activity was demonstrated in cell culture by the Nb2 proliferation assay.

[Anti-oxidative effect of ribonuclease inhibitor by site-directed mutagenesis and expression in Pichia pastoris].

Human placental ribonuclease inhibitor is an acidic protein of Mr approximately 50 kDa with unusually high contents of leucine and cysteine. It is a cytosolic protein that protects cells from the adventitious invasion of pancreatic-type ribonuclease. HRI has 32 cysteine residues, and the oxidative formation of disulfide bonds from those cysteine residues is a rapid cooperative process that inactivates HRI. The most proximal cysteine residues in native HRI are two pairs that are adjacent in sequence. In the present paper, two molecules of alanine to substitute for cys328/cys329 were performed by site-directed mutagenesis. The site-mutated RI cDNA was constructed into plasmid pPIC9K, and then transformed Pichia pastoris GS115 by electroporation. After colony screening , the bacterium was cultured and the product was purified with affinity chromatography. The affinity of the recombinant human RI with double site mutation was examined for RNase A and its anti-oxidative effect. The results indicated that there was no much change in the affinity for RNase A detected when compared with the wild type of RI. But the capacity of anti-oxidative effect was increased by 7-9 times. The enhance in anti-oxidative effect might be the reason for preventing the formation of disulfide bond between cys328 and cys329 and the three dimensional structure of RI was thereby maintained.

Antitumor effects of human ribonuclease inhibitor gene transfected on B16 melanoma cells.

Human ribonuclease inhibitor (RI) is a cytoplasmic acidic protein. The experiment demonstrated that it might effectively inhibit tumor-induced angiogenesis and inhibit tumor growth. Ribonuclease inhibitor is constructed almost entirely of leucine-rich repeats, which might be involved in unknown biological effects besides inhibiting RNase A and angiogenin activities. The exact molecular mechanism of antitumor on ribonuclease inhibitor remains unclear so far. In order to further understand the function of ribonuclease inhibitor and investigate the relationship with tumor growth, our study established a transfection of human ribonuclease inhibitor cDNA into the murine B16 cells by the retroviral packaging cell line PA317. The cell line transfected with a stably high expression of ribonuclease inhibitor was identified. We found that the transfected ribonuclease inhibitor could obviously inhibit cell proliferation, regulate cell cycle and induce cell apoptosis in vitro. Mice that were injected with the B16 cells transfected RI cDNA showed a significant inhibition of the tumor growth with lighter tumor weight, lower density of microvessels, longer latent periods, and survival time than those in the other two control groups. In conclusion, the results reveal the novel mechanism that antitumor effect of ribonuclease inhibitor is also associated with inducing apoptosis, regulating cell cycle and inhibiting proliferation besides antiangiogenesis. These results suggest that ribonuclease inhibitor might be a candidate of tumor suppressor gene in some tissues. RI could become a target gene for gene therapy. Our study may be of biological and clinical importance.

Stimulation of human trophoblast invasion by placental growth hormone.

A critical step in establishment of human pregnancy is the invasion of the uterus wall by the extravillous cytotrophoblast (EVCT), a process regulated by multiple autocrine and paracrine factors. Hormones belonging to the GH/prolactin family are expressed at the maternofetal interface. Because they are involved in cell motility in various models, we examined the possible regulatory role of human placental GH (hPGH) in EVCT invasiveness. By using an in vitro invasion model, we found that EVCT isolated from first-trimester chorionic villi and cultured on Matrigel secreted hPGH and expressed human GH receptor (hGHR). These data were confirmed by in situ immunohistochemistry. EVCT expressed the full-length and truncated forms of hGHR, and the Janus kinase-2/signal transducer and activator of transcription factor-5 signaling pathway was activated in EVCT by hPGH treatment. Strong hPGH and hGHR expression was observed when EVCT invaded Matrigel and moved through the pores of the filter on which they were cultured. hPGH stimulated EVCT invasiveness, and this effect was inhibited by a Janus kinase-2 inhibitor. Interestingly, hPGH was more efficient than pituitary GH in stimulating EVCT invasiveness. These results offer the first evidence for a placental role of hPGH and suggest an autocrine/paracrine role of hPGH in the regulation of trophoblast invasion.

[Expression of a human ribonuclease inhibitor variant in Escherichia coli and silkworm insect cell (Bombyx mori)].

Ribonuclease inhibitor (RI) is an acidic 50 kD protein with a high content of leucine and cysteine residues. RI inhibits RNases of the pancreatic type. A variant of RI was cloned from human fetal liver cDNA library by polymerase chain reaction (PCR). Compared with the reported RI, only two variations Arg(359)Ala and Leu(365)Pro were found in RIv amino acid sequence. Recombinant RIv has been expressed both in Escherichia coli and silkworm insect cells (Bombyx mori). The recombinant RIv exhibited inhibition activity on ribonucleolytic activity of RNase A in vitro system.

Ovine placental lactogen specifically binds to endometrial glands of the ovine uterus.

A hormonal servomechanism has been proposed to regulate differentiation and function of the endometrial glandular epithelium (GE) in the ovine uterus during pregnancy. This mechanism involves sequential actions of estrogen, progesterone, ovine interferon tau (IFNtau), placental lactogen (oPL), and placental growth hormone (oGH). The biological actions of oPL in vitro are mediated by homodimerization of the prolactin receptor (oPRLR) and heterodimerization of the oPRLR and oGH receptor. The objectives of the study were to determine the effects of intrauterine oPL, oGH, and their combination on endometrial histoarchitecture and gene expression and to localize and characterize binding sites for oPL in the ovine uterus in vivo using an in situ ligand binding assay. Intrauterine infusion of oPL and/or oGH following IFNtau into ovariectomized ewes treated with progesterone daily differentially affected endometrial gland number and expression of uterine milk proteins and osteopontin. However, neither hormone affected PRLR, insulin-like growth factor (IGF)-I, or IGF-II mRNA levels in the endometrium. A chimeric protein of placental secretory alkaline phosphatase (SEAP) and oPL was used to identify and characterize binding sites for oPL in frozen sections of interplacentomal endometrium from pregnant ewes. Specific binding of SEAP-oPL was detected in the endometrial GE on Days 30, 60, 90, and 120 of pregnancy. In Day 90 endometrium, SEAP-oPL binding to the endometrial GE was displaced completely by oPL and prolactin (oPRL) but only partially by oGH. Binding experiments using the extracellular domain of the oPRLR also showed that iodinated oPL binding sites could be competed for by oPRL and oPL but not by oGH. Collectively, results indicate that oPL binds to receptors in the endometrial glands and that oPRL is more effective than oGH in competing for these binding sites. Thus, effects of oPL on the endometrial glands may be mediated by receptors for oPRL and oGH.

Human placental growth hormone causes severe insulin resistance in transgenic mice.

OBJECTIVE: The insulin resistance of pregnancy is considered to be mediated by human placental lactogen, but the metabolic effects of human placental growth hormone have not been well defined. Our aim was to evaluate the effect of placental growth hormone on insulin sensitivity in vivo using transgenic mice that overexpress the human placental growth hormone gene. STUDY DESIGN: Glucose and insulin tolerance tests were performed on 5 transgenic mice that overexpressed the human placental growth hormone variant gene and 6 normal littermate controls. The body composition of the mice was assessed by dual-energy radiograph absorptiometry, and free fatty acid levels were measured as a marker of lipolysis. RESULTS: The human placental growth hormone levels in the transgenic mice were comparable to those attained in the third trimester of pregnancy. These mice were nearly twice as heavy as the control mice, and their body composition differed by a significant increase in bone density and a small decrease in percentage of body fat. Fasting insulin levels in the transgenic mice that overexpressed placental growth hormone were approximately 4-fold higher than the control mice (1.57 +/- 0.22 ng/mL vs 0.38 +/- 0.07 ng/mL; P <.001) and 7 times higher 30 minutes after glucose stimulation (4.17 +/- 0.54 ng/mL vs 0.62 +/- 0.10 ng/mL; P <.0001) with no significant difference in either fasting or postchallenge glucose levels. Insulin sensitivity was markedly decreased in the transgenic mice, as demonstrated by an insignificant decline in glucose levels after insulin injection compared with the control mice, which demonstrated more than a 65% reduction in glucose levels (P <.001). CONCLUSION: Human placental growth hormone causes insulin resistance as manifested by fasting and postprandial hyperinsulinemia and minimal glucose lowering in response to insulin injection. Human placental growth hormone is a highly likely candidate to mediate the insulin resistance of pregnancy.

Preparation of potent cytotoxic ribonucleases by cationization: enhanced cellular uptake and decreased interaction with ribonuclease inhibitor by chemical modification of carboxyl groups.

Carboxyl groups of bovine RNase A were amidated with ethylenediamine (to convert negative charges of carboxylate anions to positive ones), 2-aminoethanol (to eliminate negative charges), and taurine (to keep negative charges), respectively, by a carbodiimide reaction. Human RNase 1 was also modified with ethylenediamine. Surprisingly, the modified RNases were all cytotoxic toward 3T3-SV-40 cells despite their decreased ribonucleolytic activity. However, their enzymatic activity was not completely eliminated by the presence of excess cytosolic RNase inhibitor (RI). As for native RNase A and RNase 1 which were not cytotoxic, they were completely inactivated by RI. More interestingly, within the cytotoxic RNase derivatives, cytotoxicity correlated well with the net positive charge. RNase 1 and RNase A modified with ethylenediamine were more cytotoxic than naturally occurring cytotoxic bovine seminal RNase. An experiment using the fluorescence-labeled RNase derivatives indicated that the more cationic RNases were more efficiently adsorbed to the cells. Thus, it is suggested that the modification of carboxyl groups could change complementarity of RNase to RI and as a result endow RNase cytotoxicity and that cationization enhances the efficiency of cellular uptake of RNase so as to strengthen its cytotoxicity. The finding that an extracellular human enzyme such as RNase 1 could be effectively internalized into the cell by cationization suggests that cationization is a simple strategy for efficient delivery of a protein into cells and may open the way of the development of new therapeutics.

Analysis of the interactions of human ribonuclease inhibitor with angiogenin and ribonuclease A by mutagenesis: importance of inhibitor residues inside versus outside the C-terminal "hot spot".

Ribonuclease inhibitor (RI) binds diverse mammalian RNases with extraordinary avidity. Here, we have investigated the structural basis for this tight binding and broad specificity by mutational analysis of the complexes of RI with angiogenin (Ang) and RNase A (K(D)=0.5 fM and 43 fM, respectively). Both crystal structures are known; the interfaces are large, and the ligands dock similarly, although few of the specific interactions formed are analogous. Our previous mutagenesis studies focused primarily on one contact region, containing RI 434-438 and the enzymatic active site. Many single-residue replacements produced extensive losses of binding energy (2.3-5.9 kcal/mol), suggesting that this region constitutes a "hot spot" in both cases. We have now explored the roles of most of the remaining RI residues that interact with Ang and/or RNase A. One major cluster in each complex lies in a Trp-rich area of RI, containing Trp261, Trp263, Trp318, and Trp375. Although the energy losses from individual replacements in this portion of the Ang complex were small-to-moderate (0-1.5 kcal/mol), the changes from multiple substitutions were much greater than additive, and the binding energy provided by this region is estimated to be approximately 6 kcal/mol (30 % of total). Effects of replacing combinations of hot spot components had also been found to be superadditive, and this negative cooperativity is now shown to extend to the neighboring contact residue RI Ser460. The overall contribution of the hot spot, taking superadditivity into account, is then approximately 14-15 kcal/mol. The hot spot and Trp-rich regions, although spatially well separated, are themselves functionally linked. No other parts of the RI-Ang interface appear to be energetically important. Binding of RNase A is more sensitive to substitutions throughout the interface, with free energy losses>/=1 kcal/mol produced by nearly all replacements examined, so that the sum of losses greatly exceeds the binding energy of the complex. This discrepancy can be explained, in part, by positive cooperativity, as evident from the subadditive effects observed when combinations of residues in either the hot spot or Trp-rich region are replaced. These findings suggest that the binding energy may be more widely distributed in the RNase A complex than in the Ang complex.