Characterization of Concanavalin A-based lectin sorbents for the extraction of the human chorionic gonadotropin glycoforms prior to analysis by nano liquid chromatography-high resolution mass spectrometry.

Human chorionic gonadotropin (hCG) is constituted of the hCGα and hCGß subunits and is a highly glycosylated protein. Affinity supports based on immobilized Concanavalin A (Con A) lectin were used in solid phase extraction (SPE) to fractionate the hCG glycoforms according to their glycosylation state. For the first time, the lectin SPE fractions were off-line analysed by a nano liquid chromatography - high-resolution mass spectrometry (nanoLC-HRMS) method keeping the glycoforms intact. For this, home-made Con A sorbents were prepared by immobilizing lectin on Sepharose with a mean grafting yield of 98.2% (relative standard deviation (RSD) of 3.5%, n = 15). A capacity of about 100 µg of purified urinary hCG (uhCG) per ml of sorbent, grafted with a density of 10 mg of Con A per ml, was estimated. Average extraction yields of around 60% for both hCGα and hCGß glycoforms were obtained after optimization of the extraction protocol. Intra- and inter-assay evaluation led to average RSD values of around 10%, indicating a repeatable extraction procedure. Similar results were obtained with commercial Con A-based sorbents but only after their 3rd use or after an extensive pre-conditioning step. Finally, the Con A SPE led to the fractionation of some glycoforms of uhCG, allowing the detection of an hCGα glycoform with two tetra-antennary N-glycans that couldn't be detected by direct analysis in nanoLC-HRMS without Con A SPE. Regarding a recombinant hCG, a fractionation was also observed leading to the detection of unretained hCGα glycoforms with tri-antennary N-glycans. Therefore, the combination of lectin SPE with intact protein analysis by nanoLC-HRMS can contribute to a more detailed glycosylation characterization of the hCG protein.

Characterization of beta subunit variants of recombinant human chorionic gonadotrophin.

Human chorionic gonadotropin (hCG), an endogenous glycoprotein hormone, has been widely used for the treatment of infertility and corpus luteum defect in women. The biological specificity of hCG is essentially determined by its beta (ß-) subunit, whereas the alpha (α-) subunit is a common subunit shared among the gonadotropin family. In development of a therapeutic recombinant hCG, the purity analysis showed that the beta (ß-) subunit has two variants, ß1 and ß2. Structural characterization using a combination of analytical techniques has demonstrated that ß1-subunit is derived from non-glycosylation at Asn 13, whereas ß2-subunit is a normal species with complete N-glycosylation at both Asn 13 and Asn 30. In vivo Bioactivity evaluation of the r-hCG fractions with various ratios of ß1-and ß2-subunits showed that incomplete glycosylation at Asn 13 potentially reduced the biological activity of r-hCG to promote uterus growth. Although hCG has a long history of medicinal use, this is the first report to identify the structural difference of hCG ß-subunit variants, as well as to preliminary establish the structure-activity relationship of this variation. The obtained results also suggest the importance of variant characterization and necessary quality control of product variants during the development of recombinant protein therapeutics.

Ag nanoparticle in situ decorated on Ti3C2Tx with excellent SERS and EIS immunoassay performance for beta-human chorionic gonadotropin.

Two-dimensional transition metal carbides, nitrides, and carbonitrides (MXene), with excellent optical and electrical properties, are promising substrates for surface-enhanced Raman scattering (SERS) and electrochemical sensors. Therefore, a unique 3D-decorated structure containing silver (Ag) nanoparticles and Ti3C2Tx was designed as the substrates of SERS and electrochemical impedance spectroscopy (EIS) immunosensors. The Ag/Ti3C2Tx composite significantly increases Raman intensity, which is attributed to the synergistic effect of Ti3C2Tx and Ag nanoparticles. Based on the SERS performance of the Ag/Ti3C2Tx composite, the magnetic properties of Fe3O4 and the specificity of antigen-antibody, a sandwich-structured SERS immunosensor is constructed, which can effectively detect trace amounts of beta-human chorionic gonadotropin (ß-hCG). The SERS immunosensor exhibits a wide linear range of 5.0 × 10-6-1.0 mIU mL-1, and a low detection limit of 9.0 × 10-7 mIU mL-1. Meanwhile, the Ag/Ti3C2Tx-based EIS immunosensor is constructed for the portable detection of ß-hCG, which exhibits a wide linear range of 5.0 × 10-2-1.0 × 102 mIU mL-1, a low detection limit of 9.5 × 10-3 mIU mL-1. Moreover, two immunosensors can be used to detect actual serum samples with satisfactory recovery (98.5-102.2%). This work could guide the design of low-cost, sensitive, flexible, and portable biosensors. The SERS and EIS substrates composited with Ti3C2Tx and Ag nanoparticles enable excellent performance for detecting ß-hCG.

Relationship between extreme values of first trimester maternal pregnancy associated plasma Protein-A, free-ß-human chorionic gonadotropin, nuchal translucency and adverse pregnancy outcomes.

OBJECTIVE: The aim of our study was to investigate the relationship between extreme values of first trimester screening markers and adverse obstetric outcomes. MATERIALS AND METHODS: Our study was conducted by examining the prenatal and postnatal perinatal records of 786 singleton gestations between the ages of 18-40, who applied to Prof. Dr. Cemil Tasçioglu City Hospital outpatient clinics for first-trimester screening for aneuploidy, between January 1, 2017 and December 31, 2019. RESULTS: The presence of small for gestational age (SGA) was found to be statistically significant for the <5 percentile (<0.37) pregnancy-associated plasma protein A (PAPP-A) group (p = 0.016). For <5 percentile ß-hCG group, the presence of gestational diabetes mellitus (GDM), premature rupture of membrane (PROM) and preterm premature rupture of membrane (PPROM) was determined as a statistically significant risk (p = 0.015, p = 0.005, p = 0.02 respectively) In the univariate test, fetal death rate was found to be high for ≥90 percentile at nuchal translucency (NT), but the presence of fetal death was found to be statistically insignificant in logistic regression analysis. (p: 0.057). CONCLUSION: First trimester screening test can be used in predicting pregnancy complications. In this study we found that serum levels of PAPP-A are associated with developing SGA, while GDM, PROM and PPROM are more common in low serum free ß-hCG.

Identification and semi-relative quantification of intact glycoforms of human chorionic gonadotropin alpha and beta subunits by nano liquid chromatography-Orbitrap mass spectrometry.

The human chorionic gonadotropin (hCG) protein belongs to a family of glycoprotein hormones called gonadotropins. It is a heterodimer made of two non-covalently linked subunits. The α-subunit structure, hCGα, has 2 N-glycosylation sites, while the beta subunit, hCGß, has 2 N- and 4 O-glycosylation sites. This leads to numerous glycoforms. A method based on the analysis of hCG glycoforms at the intact level by nano-reversed phase liquid chromatography coupled to high resolution mass spectrometry (nanoLC-HRMS) with an Orbitrap analyzer was previously developed using a recombinant hCG-based drug, Ovitrelle®, as standard. It allowed the detection of about 30 hCGα glycoforms, but didn't allow the detection of hCGß glycoforms. This method was thus here significantly modified (addition of a pre-concentration step of the sample to increase the sample volume from 70 nl to 1 µl, optimization of the gradient slope and the nature and content of the acidic additive in the mobile phase). It led to an improvement of the separation of hCGα and hCGß glycoforms, which allowed for the first time the detection of 33 hCGß glycoforms at intact level. In addition, a higher number of hCGα glycoforms (42 in total, i.e. a 40% increase) was detected. The figures of merit of this new method were next assessed. The relative standard deviations (RSDs) of the retention time ranged between 0.02 and 0.95% (n = 3), with an average value of 0.36% for the alpha glycoforms and between 0.01 and 1.08% (n = 3) with an average value of 0.23% for the beta glycoforms. The RSDs of the relative peak area measured on the extracted ion chromatogram of each glycoform were below 20% (n = 3), with an average value of 9.8%, thus allowing semi-relative quantification. Therefore, this method has a high potential for rapid quality control aiming for the detection and comparison of glycoforms present in glycoprotein-based pharmaceutical preparations.

N- and O-glycosylation patterns and functional testing of CGB7 versus CGB3/5/8 variants of the human chorionic gonadotropin (hCG) beta subunit.

The classical function of human chorionic gonadotropin (hCG) is its role in supporting pregnancy. hCG is a dimer consisting of two highly glycosylated subunits, alpha (CGA) and beta (CGB). The beta-hCG protein is encoded by CGB3, CGB5, CGB7 and CGB8 genes. CGB3, 5 and 8 code for an identical protein, CGB3/5/8, whereas CGB7 differs in three amino acids from CGB3/5/8. We had observed earlier that CGB7 and CGB3/5/8 display very distinct tissue expression patterns and that the tumor suppressor and transcription factor p53 can activate expression of CGB7 but not of CGB3/5/8 genes. Here, we investigate the glycan structures and possible functional differences of the two CGB variants. To this end, we established a system to produce and isolate recombinant CGA, CGB7 and CGB3/5/8 proteins. We found that N- and O-glycosylation patterns of CGB7 and CGB3/5/8 are quite similar. Functional assays were performed by testing activation of the ERK1/2 pathway and demonstrated that CGB7 and CGB5/5/8 appear to be functionally redundant isoforms, although a slight difference in the kinetics of ERK1/2 pathway activation was observed. This is the first time that biological activity of CGB7 is shown. In summary, the results lead to the hypothesis that CGB7 and CGB3/5/8 do not hold significant functional differences but that timing and cell type of their expression is the key for understanding their divergent evolution.

Unbalanced human embryos secrete more hyperglycosylated human chorionic gonadotrophin (hCG-H) than balanced ones.

PURPOSE: The aim of this study was to compare the levels of hyperglycosylated human chorionic gonadotropin (hCG-H) secreted from balanced and unbalanced human embryos. METHODS: Single-step culture media samples from 155 good quality embryos, derived from 90 good prognosis patients undergoing intracytoplasmic sperm injection (ICSI), were collected on the fifth day of embryo cultivation. All embryos were tested by next-generation sequencing (NGS) technique. The hCG-H levels in the culture media were evaluated by ELISA kit (Cusabio Biotech, CBS-E15803h) according to the manufacturer's instructions. Statistical analysis was performed using SPSS v.21 (IBM Corp., Armonk, NY, USA). RESULTS: The NGS analysis revealed that 36% of the embryos (n = 56) were balanced, and 64% of the embryos were unbalanced (n = 99). The presence of hCG-H was confirmed in all embryo culture media samples but was absent in the negative control. In addition, hCG-H concentration was significantly higher in the culture media from unbalanced embryos compared with the balanced ones (0.72 ± 0.30 mIU/ml vs. 0.62 ± 0.12 mIU/ml, p = 0.02, respectively). Furthermore, the mean levels of hCG-H were significantly increased in the samples from embryos with multiple abnormalities. Finally, the highest levels of hCG-H were expressed from embryos with monosomy of chromosome 11 (1.28 ± 0.04 mIU/ml) and those with trisomies of chromosomes 21 (2.23 mIU/ml) and 4 (1.02 ± 0.35 mIU/ml). CONCLUSION: Our results suggest that chromosomal aberrations in human embryos are associated with an increased secretion of hCG-H. However, hCG-H concentration in embryo culture media as a single biomarker is not sufficient for an accurate selection of balanced embryos.

Development and biological activity of long-acting recombinant human interferon-α2b.

BACKGROUND: The type I human interferon (IFN) family consists of a group of cytokines with a multiplicity of biological activities, including antiviral, antitumor, and immunomodulatory effects. However, because the half-life of IFN is short, its clinical application is limited. Increasing the yield and biological activity of IFN while extending its half-life is currently the focus of IFN research. RESULTS: Two novel long-acting recombinant human IFN-α2b (rhIFN-α2b) proteins were designed in which the carboxyl-terminal peptide (CTP) of the human chorionic gonadotropin ß su bunit and N-linked glycosylation sequences were linked to rhIFN-α2b. They were designated IFN-1CTPON (fused at the C-terminus of rhIFN-α2b) and IFN-2CTPON (fused at both the C-terminus and N-terminus of rhIFN-α2b). Monoclonal CHO cell strains stably and efficiently expressing the IFNs were successfully selected with methotrexate (MTX), and the highest expression levels were 1468 mg/l and 1196 mg/l for IFN-1CTPON and IFN-2CTPON, respectively. The proteins were purified with affinity chromatography and molecular sieve chromatography. IFN-1CTPON and IFN-2CTPON showed antiviral and antiproliferative activities in vitro. Notably, the half-life of IFN-1CTPON and IFN-2CTPON in vivo were three-fold and two-fold longer than that of commercially available rhIFN-α2b. CONCLUSIONS: CHO cell strains stably expressing long-acting rhIFN-α2b were screened. The purified IFN-CTPON protein has biological activity and an extended half-life, and therefore potential applications.

Urinary human chorionic gonadotropin isoform concentrations in doping control samples.

Anti-doping laboratories routinely use immunoassays to measure urinary concentrations of human chorionic gonadotropin (hCG). To minimize immunoassay differences and false positive screen results from inactive isoforms (free ß-subunit (hCGß), ß-subunit core fragment (hCGßcf)) laboratories now use intact hCG instead of total hCG immunoassays to measure hCG. To determine the distribution of hCG isoforms in urine, we determined the concentrations of intact hCG, hCGß, and hCGßcf in male urine samples based on immunoassay total hCG concentrations using a sequential immunoextraction and a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. hCG was isolated using antibody-conjugated magnetic beads and unique tryptic peptides were quantified by LC-MS/MS. Negative samples with detectable but low total hCG concentrations (1.2-3.5 pmol/L) had intact and hCGß concentrations <1.2 pmol/L, and hCGßcf concentrations <2.3 pmol/L by LC-MS/MS. Urine samples from an athlete receiving hCG had intact hCG concentrations ranging from 18.8 to 57.6 pmol/L, hCGß concentrations <0.7 pmol/L, and hCGßcf concentrations ranging from 94 to 243% of the intact hCG concentration. In 27 atypical samples with total hCG concentrations ranging from 16.7 to 412.7 pmol/L with intact hCG <2.7 pmol/L by immunoassay, all samples had intact hCG concentrations <3.8 pmol/L and hCGß concentrations <6.2 pmol/L by LC-MS/MS. hCGßcf concentrations by LC-MS/MS varied widely and ranged from 1.03 to 21.9 pmol/L. In summary, total hCG immunoassays significantly overestimate hCG concentrations and can produce false positive results. Although the intact hCG immunoassay slightly overestimates hCG concentrations compared to LC-MS/MS, it can distinguish between cases of hCG use and atypical cases with elevated total hCG concentrations. Copyright © 2016 John Wiley & Sons, Ltd.

Improvement of in vitro stability and pharmacokinetics of hIFN-α by fusing the carboxyl-terminal peptide of hCG ß-subunit.

Improving in vivo half-life and in vitro stability of protein-based therapeutics is a current challenge for the biopharmaceutical industry. In particular, recombinant human interferon alpha-2b (rhIFN-α2b), which belongs to a group of cytokines extensively used for the treatment of viral diseases and cancers, shows a poor stability in solution and an extremely short plasma half-life which determines a strict therapeutic regimen comprising high and repeated doses. In this work, we have used a strategy based on the fusion of the carboxyl-terminal peptide (CTP) of human chorionic gonadotropin (hCG) ß-subunit, bearing four O-linked oligosaccharide recognition sites, to each or both N- and C-terminal ends of rhIFN-α2b. Molecules containing from 5 (CTP-IFN and IFN-CTP) to 9 (CTP-IFN-CTP) O-glycosylation sites were efficiently expressed and secreted to CHO cells supernatants, and exhibited antiviral and antiproliferative bioactivities in vitro. Significant improvements in pharmacokinetics in rats were achieved through this approach, since the doubly CTP-modified IFN variant showed a 10-fold longer elimination half-life and a 19-fold decreased plasma apparent clearance compared to the wild-type cytokine. Moreover, CTP-IFN-CTP demonstrated a significant increase in in vitro thermal resistance and a higher stability against plasma protease inactivation, both features attributed to the stabilizing effects of the O-glycans provided by the CTP moiety. These results constitute the first report that postulates CTP as a tag for improving both the in vitro and in vivo stability of rhIFN-α2b which, in turn, would positively influence its in vivo bioactivity.

Oligopeptides of Chorionic Gonadotropin ß-Subunit in Induction of T Cell Differentiation into Treg and Th17.

The role of oligopeptides of chorionic gonadotropin ß-subunit (LQGV, AQGV, and VLPALP) in induction of differentiation into T-regulatory lymphocytes (Treg) and IL-17-producing lymphocytes (Th17) was studied in an in vitro system. Chorionic gonadotropin and oligopeptides promoted CD4(+) cell differentiation into functionally active Treg (FOXP3(+)GITR(+) and FOXP3(+)CTLA-4(+)), while chorionic gonadotropin and AQGV additionally stimulated IL-10 production by these cells. In parallel, chorionic gonadotropin and oligopeptides prevented CD4(+) cell differentiation into Th17 lymphocytes (ROR-gt(+)IL-17A(+)) and suppressed IL-17A secretion. Hence, oligopeptides of chorionic gonadotropin ß-subunit promoted differentiation of CD4(+) cells into Treg and, in parallel, suppress Th17 induction, thus virtually completely reproducing the effects of the hormone, which opens new vista for their use in clinical practice.

The recombinant equine LHß subunit combines divergent intracellular traits of human LHß and CGß subunits.

The pituitary LHß and placental CGß subunits are products of different genes in primates. The major structural difference between the two subunits is in the carboxy-terminal region, where the short carboxyl sequence of hLHß is replaced by a longer O-glycosylated carboxy-terminal peptide in hCGß. In association with this structural deviation, there are marked differences in the secretion kinetics and polarized routing of the two subunits. In equids, however, the CGß and LHß subunits are products of the same gene expressed in the placenta and pituitary (LHß), and both contain a carboxy-terminal peptide. This unusual expression pattern intrigued us and led to our study of eLHß subunit secretion by transfected Chinese hamster ovary and Madin-Darby canine kidney cells. In continuous labeling and pulse-chase experiments, the secretion of the eLHß subunit from the transfected Chinese hamster ovary cells was inefficient (medium recovery of 16%-25%) and slow (t1/2 > 6.5 hours). This indicated that, the secretion of the eLHß subunit resembles that of hLHß rather than hCGß. In Madin-Darby canine kidney cells grown on Transwell filters, the eLHß subunit was preferentially secreted from the apical side, similar to the hCGß subunit secretory route (∼65% of the total protein secreted). Taken together, these data suggested that secretion of the eLHß subunit integrates features of both hLHß and hCGß subunits. We propose that the evolution of this intracellular behavior may fulfill the physiological demands for biosynthesis of the LH and CG ß-subunits in the pituitary and placenta, respectively.

Direct analysis of hCGßcf glycosylation in normal and aberrant pregnancy by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The analysis of human chorionic gonadotropin (hCG) in clinical chemistry laboratories by specific immunoassay is well established. However, changes in glycosylation are not as easily assayed and yet alterations in hCG glycosylation is associated with abnormal pregnancy. hCGß-core fragment (hCGßcf) was isolated from the urine of women, pregnant with normal, molar and hyperemesis gravidarum pregnancies. Each sample was subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis following dithiothreitol (DTT) reduction and fingerprint spectra of peptide hCGß 6-40 were analyzed. Samples were variably glycosylated, where most structures were small, core and largely mono-antennary. Larger single bi-antennary and mixtures of larger mono-antennary and bi-antennary moieties were also observed in some samples. Larger glycoforms were more abundant in the abnormal pregnancies and tri-antennary carbohydrate moieties were only observed in the samples from molar and hyperemesis gravidarum pregnancies. Given that such spectral profiling differences may be characteristic, development of small sample preparation for mass spectral analysis of hCG may lead to a simpler and faster approach to glycostructural analysis and potentially a novel clinical diagnostic test.

Chemical synthesis of the ß-subunit of human luteinizing (hLH) and chorionic gonadotropin (hCG) glycoprotein hormones.

Human luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) are human glycoprotein hormones each consisting of two subunits, an identical α-subunit and a unique ß-subunit, that form noncovalent heterodimers. Structurally, ß-hCG shares a high degree of sequence similarity with ß-hLH, including a common N-glycosylation site at the N-terminus but differs mainly in the presence of an extended C-terminal portion incorporating four closely spaced O-linked glycans. These glycoproteins play important roles in reproduction and are used clinically in the treatment of infertility. In addition, the role of hCG as a tumor marker in a variety of cancers has also attracted significant interest for the development of cancer vaccines. In clinical applications, these hormones are administered as mixtures of glycoforms due to limitations of biological methods in producing homogeneous samples of these glycoproteins. Using the powerful tools of chemical synthesis, the work presented herein focuses on the highly convergent syntheses of homogeneous ß-hLH and ß-hCG bearing model glycans at all native glycosylation sites. Key steps in these syntheses include a successful double Lansbury glycosylation en route to the N-terminal fragment of ß-hCG and the sequential installation of four O-linked glycosyl-amino acid cassettes into closely spaced O-glycosylation sites in a single, high-yielding solid-supported synthesis to access the C-terminal portion of the molecule. The final assembly of the individual glycopeptide fragments involved a stepwise native chemical ligation strategy to provide the longest and most complex human glycoprotein hormone (ß-hCG) as well as its closely related homologue (ß-hLH) as discrete glycoforms.

The role of the 3' region of mammalian gonadotropin ß subunit gene in the luteinizing hormone to chorionic gonadotropin evolution.

CGß subunits comprise a unique carboxyl-terminal peptide (CTP) that has multiple O-linked glycans and extends serum half-life of the protein. It has evolved by incorporating a previously untranslated region of the LHß gene into the reading frame. Although CTP-like sequences are encrypted in the LHß genes of several mammals, the CGß subunit developed only in primates and equids. To study this restriction in evolution, we examined whether the cryptic CTP decoded from the bovine LHß gene (boCTP) possesses key characteristics of the human (h) CGß-CTP. The boCTP does not impede several crucial aspects of hormone biosynthesis, but compared to the hCGß-CTP, the stretch lacks O-glycans and determinants for circulatory survival. O-glycan deficiency and the associated incapacity to extend serum half-life is a major drawback of the boCTP. This may explain why LH did not evolve into CG in ruminants and consequently alternative mechanisms evolved to delay luteolysis early in gestation.

Structural and functional analysis of rare missense mutations in human chorionic gonadotrophin ß-subunit.

Heterodimeric hCG is one of the key hormones determining early pregnancy success. We have previously identified rare missense mutations in hCGß genes with potential pathophysiological importance. The present study assessed the impact of these mutations on the structure and function of hCG by applying a combination of in silico (sequence and structure analysis, molecular dynamics) and in vitro (co-immunoprecipitation, immuno- and bioassays) approaches. The carrier status of each mutation was determined for 1086 North-Europeans [655 patients with recurrent miscarriage (RM)/431 healthy controls from Estonia, Finland and Denmark] using PCR-restriction fragment length polymorphism. The mutation CGB5 p.Val56Leu (rs72556325) was identified in a single heterozygous RM patient and caused a structural hindrance in the formation of the hCGα/ß dimer. Although the amount of the mutant hCGß assembled into secreted intact hCG was only 10% compared with the wild-type, a stronger signaling response was triggered upon binding to its receptor, thus compensating the effect of poor dimerization. The mutation CGB8 p.Pro73Arg (rs72556345) was found in five heterozygotes (three RM cases and two control individuals) and was inherited by two of seven studied live born children. The mutation caused ~50% of secreted ß-subunits to acquire an alternative conformation, but did not affect its biological activity. For the CGB8 p.Arg8Trp (rs72556341) substitution, the applied in vitro methods revealed no alterations in the assembly of intact hCG as also supported by an in silico analysis. In summary, the accumulated data indicate that only mutations with neutral or mild functional consequences might be tolerated in the major hCGß genes CGB5 and CGB8.

Antibody recognition of a human chorionic gonadotropin epitope (hCGbeta66-80) depends on local structure retained in the free peptide.

Human chorionic gonadotropin (hCG) is an important biomarker in pregnancy and oncology, where it is routinely detected and quantified by specific immunoassays. Intelligent epitope selection is essential to achieving the required assay performance. We present binding affinity measurements demonstrating that a typical ß3-loop-specific monoclonal antibody (8G5) is highly selective in competitive immunoassays and distinguishes between hCGß(66-80) and the closely related luteinizing hormone (LH) fragment LHß(86-100), which differ only by a single amino acid residue. A combination of optical spectroscopic measurements and atomistic computer simulations on these free peptides reveals differences in turn type stabilized by specific hydrogen bonding motifs. We propose that these structural differences are the basis for the observed selectivity in the full protein.

Surface-enhanced Raman scattering and theoretical studies of the C-terminal peptide of the ß-subunit human chorionic gonadotropin without linked carbohydrates.

Raman and surface-enhanced Raman scattering (SERS) spectra of the synthetic carboxy terminal peptide of human chorionic gonadatropin ß-subunit free of carbohydrate moieties(P37) are reported. The spectral analysis is performed on the basis of our reported Raman spectrum and SERS data of oligopeptides displaying selected amino acids sequences MRKDV, ADEDRDA, and LGRGISL. SERS samples of P37 were prepared by coating the solid peptide with metal colloids on a quartz slide. This treatment makes possible to obtain high spectral batch to batch reproducibility. Amino acids components of P37 display net charges and hydrophobic characteristics, which are related to particular structural aspects of the adsorbate-substrate interaction. The spectroscopic results are supported by quantum chemical calculations performed by using extended Hückel theory method for a model of P37 interacting with an Ag surface. The P37-metal interaction is drove by positively charged fragments of selected amino acids,mainly threonine 109, lysine 122, and arginine in positions 114 and 133. Data here reported intend to contribute to the knowledge about the antigen-antibody interaction and to the drugs delivery research area

Immunogenic comparison for two different recombinant chimeric peptides (CP12 and CP22) containing one or two copies of three linear B cell epitopes from ß-hCG subunit.

To develop a superior chimeric peptide (CP) vaccine of human chorionic gonadotropin (hCG), two CP antigens (named CP12 and CP22) encoding one or two copies of three linear B cell epitopes from the ß-hCG subunit and six foreign T cell epitopes, including two promiscuous TCEs from hepatitis B surface antigen and tetanus toxoid, were constructed and biosynthesized. The hCG CP12 and CP22 of 21 or 23 kDa, respectively, were expressed in Escherichia coli at the level of ~1% of total cell proteins when inserted into thermo-inducible pBV221 expression vector. The purified CP12 and CP22 proteins with >95% relative homogeneity are immunogenic, and elicited antibodies against the ß5, ß9 and ß8 BCEs of ß-hCG in both rabbits and three different inbred strains of mice. A mouse uterine weight study in Balb/c mice demonstrated that the CP12 and CP22 antigens with an additional ß5 neutralizing epitope enhanced the in vivo bio-neutralization capacity of the induced antibodies compared to the C-terminal immunogen of ß-hCG. We propose that the biosynthesized CP22, possessing with two copies of three BCEs, represents a novel candidate antigen for an hCG contraceptive or tumor therapeutic vaccine.

Designing a long-acting human growth hormone (hGH) by fusing the carboxyl-terminal peptide of human chorionic gonadotropin beta-subunit to the coding sequence of hGH.

Chimeric genes were constructed by fusing of human GH (hGH) cDNA to one, two, or three cassettes of the carboxyl-terminal peptide (CTP) of human chorionic gonadotropin (hCG)-beta-subunit. hGH variant genes were inserted into the pCI-DHFR plasmid, transfected into DG44 cells, and stable clones were selected. Bioactivity and pharmacokinetic studies were performed in hypophysectomized Sprague Dawley derived male rats. The results indicated that sc injections of GH-wild-type (WT), Biotropin (commercial), GH-CTP, or CTP-GH (0.6 mg/kg) once every 5 d for 11 d (total dose of 1.2 mg/kg) resulted in an increased weight gain by 4, 4.9, 5.1, and 7 g, respectively. Treatment with CTP-GH-CTP-CTP (GH-LA) or CTP-GH-CTP (0.6 mg/kg) once every 5 d for 11 d or with Biotropin (0.12 mg/kg) daily for 11 d (total dose 1.2 mg/kg) resulted in a dramatic increase in weight gain of 16.5, 16.8, and 17 g, respectively. Repeated injections with different doses of GH-LA, 0.6, 1.8 mg/kg every 4 d or daily injection of 0.12 mg/kg of Biotropin increased the weight gain by 16, 28, and 18 gr, respectively. In addition, the cumulative serum levels of IGF-I after injection of GH-LA was significantly higher than that detected after injection of Biotropin. Pharmacokinetic studies indicated that the half-life, mean residence time, area under the curve, time of maximal plasma concentration, and maximal plasma concentration of GH-LA are dramatically increased compared with Biotropin. This may suggest that the mechanism of GH metabolic clearance is affected by the presence of CTP. These data establish a rationale for using this chimera as a long-acting GH analog.