Analysis of the human chorionic gonadotropin protein at the intact level by HILIC-MS and comparison with RPLC-MS.

In the present work, the human chorionic gonadotropin (hCG) hormone was characterized for the first time by hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution (HR) quadrupole/time-of-flight (qTOF) mass spectrometry (MS) at the intact level. This heterodimeric protein, consisting of two subunits (hCGα and hCGß), possesses 8 potential glycosylation sites leading to a high number of glycoforms and has a molecular weight of about 35 kDa. The HILIC conditions optimized in a first paper but using UV detection were applied here with MS for the analysis of two hCG-based drugs, a recombinant hCG and a hCG isolated from the urine of pregnant women. An amide column (150 × 2.1 mm, 2.6 µm, 150 Å), a mobile phase composed of acetonitrile and water both containing 0.1% of trifluoroacetic acid, and a temperature of 60 °C were used. The gradient was from 85 to 40% ACN in 30 min. The use of TFA that had been shown to be necessary for the separation of glycoforms caused, as expected, an ion suppression effect in MS that was partially overcome by increasing the amount of protein injected (2 µL at 1 mg mL-1) and reducing the detection m/z range (from 1500 to 300). These conditions allowed the detection of different glycoforms of hCGα. The performance of the HILIC-HRMS method was compared with that previously obtained in RPLC-HRMS in terms of the number of detected glycoforms, selectivity, and sensitivity. The complementarity and orthogonality of the HILIC and RP modes for the analysis of hCG at the intact level were demonstrated.

[Gangliocytic paragangliomas: a clinicopathologic study].

Objective: To investigate the clinicopathological features of gangliocytic paraganglioma(GP). Methods: Clinical data and pathological diagnosis of the 4 cases of GP were obtained through the medical record inquiry from January 2011 to December 2017 at the First Affiliated Hospital of Zhengzhou University. Routine HE staining and immunohistochemistry of CKpan, Syn, CgA, CD56, NSE and NF were performed. Clinical follow-up of the patients was obtained through telephone communication. Results: All 4 patients, including 2 male and 2 female patients, presented with intermittent abdominal pain and distention. The median age was 56 years. Preoperative CT showed local thickening of the duodenum wall with slight enhancement in all four cases. Endoscopic ultrasonography showed low level echo in the mucous layer and submucosa involved by the tumor in 3 of 4 cases. The maximal diameter of the tumor ranged from 0.6 to 1.8 cm with an average of 1.2 cm. Microscopically, the tumors consisted of epithelioid, spindle and ganglion-like cells, and the proportion of the three cell types was different among cases. Epithelioid cells expressed CKpan, Syn, CgA and CD56. Spindle cells expressed S-100 protein and SOX-10 and ganglion-like cells expressed NF, Syn, CgA and CD56.All tumour cells expressed NSE. All 4 patients had no recurrence a post-surgery follow-up period of 3 to 30 months. Conclusions: GP of the duodenum is a benign tumor with excellent prognosis after endoscopic excision. Although its incidence is very low, its diagnosis should be considered for any mass lesion of the duodenum, especially involving mucosa and submucosa of the second dudenal segment.

Machine learning to improve false-positive results in the Dutch newborn screening for congenital hypothyroidism.

OBJECTIVE: The Dutch Congenital hypothyroidism (CH) Newborn Screening (NBS) algorithm for thyroidal and central congenital hypothyroidism (CH-T and CH-C, respectively) is primarily based on determination of thyroxine (T4) concentrations in dried blood spots, followed by thyroid-stimulating hormone (TSH) and thyroxine-binding globulin (TBG) measurements enabling detection of both CH-T and CH-C, with a positive predictive value (PPV) of 21%. A calculated T4/TBG ratio serves as an indirect measure for free T4. The aim of this study is to investigate whether machine learning techniques can help to improve the PPV of the algorithm without missing the positive cases that should have been detected with the current algorithm. DESIGN & METHODS: NBS data and parameters of CH patients and false-positive referrals in the period 2007-2017 and of a healthy reference population were included in the study. A random forest model was trained and tested using a stratified split and improved using synthetic minority oversampling technique (SMOTE). NBS data of 4668 newborns were included, containing 458 CH-T and 82 CH-C patients, 2332 false-positive referrals and 1670 healthy newborns. RESULTS: Variables determining identification of CH were (in order of importance) TSH, T4/TBG ratio, gestational age, TBG, T4 and age at NBS sampling. In a Receiver-Operating Characteristic (ROC) analysis on the test set, current sensitivity could be maintained, while increasing the PPV to 26%. CONCLUSIONS: Machine learning techniques have the potential to improve the PPV of the Dutch CH NBS. However, improved detection of currently missed cases is only possible with new, better predictors of especially CH-C and a better registration and inclusion of these cases in future models.

[First-trimester screening for trisomies 18 and 13 with the combined use of the risk algorithms for trisomy 21, 18 and 13]. / Screening auf Trisomie 18 und Trisomie 13 durch das kombinierte Anwenden der Risiko-Algorithmen für Trisomie 21, 18 und 13.

PURPOSE: Assessment of first-trimester combined screening for trisomy 18 and 13 with the combined use of the risk algorithms for trisomy 21, 18 and 13. MATERIALS AND METHODS: First-trimester combined screening based on maternal and gestational age, fetal NT, PAPP-A and free ß-hCG was assessed in 39 ,004 pregnancies. Patient-specific risks for trisomy 21, 18, 13 were computed based on the current FMF London algorithm. RESULTS: The study population consisted of 38 ,751 singleton pregnancies including 39 cases with trisomy 18 or 13. In the aneuploid group, median delta NT was 0.72 mm, PAPP-A was 0.21 MoM and free ß-hCG was 0.33 MoM. Although only 41 % of the NT measurements of fetuses with trisomy 18 or 13 were above the 95th percentile, the detection rates for trisomy 18 or 13 were 82 % with the trisomy 18/13 algorithm and 56.4 % with the trisomy 21 algorithm. The respective false-positive rates were 0.7 % and 4.7 %. The combination of the trisomy 18/13 and the trisomy 21 algorithm with the same cut-offs led to a detection rate of 94.9 % at an overall false-positive rate of 5.0 %. CONCLUSION: Despite a substantial underestimation of the fetal NT, the combined use of the trisomy 18/13 and the trisomy 21 algorithm of the FMF London leads to a detection rate for trisomy 18/13 of about 95 % for a false-positive rate of 5.0 %.

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.

Pituitary Glycoprotein Hormones in Human Milk before and after Pasteurization or Refrigeration.

Our aims were to investigate the presence of pituitary glycoprotein hormones in preterm and donor milk, and to examine the effects of Holder pasteurization and refrigeration on the levels of these hormones. We measured follicle-stimulating hormone (FSH), luteinizing hormone (LH), and thyroid-stimulating hormone (TSH) in milk samples from mothers who delivered prematurely (n = 27) and in samples of mothers who delivered at term and donated milk to the Mother's Milk Bank of Iowa (n = 30). The gonadotropins and TSH were present in similar amounts within human milk produced for preterm and term infants. FSH increased 21% after refrigeration (p < 0.05), while LH declined by 39% (p < 0.05). Holder pasteurization decreased LH by 24% (p < 0.05) and increased TSH by 17% (p < 0.05). Holder pasteurization followed by refrigeration resulted in a 21% increase in FSH and a 41% decrease in LH (both p < 0.05), resulting in more than a 3-fold increase in donor milk FSH:LH ratios (p < 0.05 versus fresh donor milk). Despite structural similarities, the gonadotropins are differentially impacted by Holder pasteurization and refrigeration, and this results in marked alterations in the relative amount of FSH and LH that may be administered to preterm infants, potentially swinging hormonal balance towards ovarian hyperstimulation in females and hypogonadism in males.

Profiling the glycoforms of the intact alpha subunit of recombinant human chorionic gonadotropin by high-resolution capillary electrophoresis-mass spectrometry.

With the rapid growth of complex heterogeneous biological molecules, effective techniques that are capable of rapid characterization of biologics are essential to ensure the desired product characteristics. To address this need, we have developed a method for analysis of intact glycoproteins based on high-resolution capillary electrophoretic separation coupled to an LTQ-FT mass spectrometer. We evaluated the performance of this method on the alpha subunit of mouse cell line-derived recombinant human chorionic gonadotrophin (r-alpha hCG), a protein that is glycosylated at two sites and is part of the clinically relevant gonadotrophin family. Analysis of r-alpha hCG, using capillary electrophoresis (CE) with a separation time under 20 min, resulted in the identification of over 60 different glycoforms with up to nine sialic acids. High-resolution CE-Fourier transform mass spectrometry (FT-MS) allowed separation and analysis of not only intact glycoforms with different numbers of sialic acids but also intact glycoforms that differed by the number and extent of neutral monosaccharides. The high mass resolution of the FT-MS enabled a limited mass range to be targeted for the examination of the protein glycoforms, simplifying the analysis without sacrificing accuracy. In addition, the limited mass range resulted in a fast scan speed that enhanced the reproducibility of the relative quantitation of individual glycoforms. The intact glycoprotein analysis was complemented with the analysis of the tryptic glycopeptides and glycans of r-alpha hCG to enable the assignment of glycan structures to individual sites, resulting in a detailed characterization of the protein. Samples of r-alpha hCG obtained from a CHO cell line were also analyzed and briefly shown to be significantly different from the murine cell line product. Taken together, the results suggest that the CE coupled to high-resolution FT-MS can be one of the effective tools for in-process monitoring as well as for final product characterization.

Aptamer-based regionally protected PCR for protein detection.

BACKGROUND: DNA aptamers are single-stranded nucleotide sequences that bind specifically to target molecules. By combining the advantages of PCR for amplifying specific DNA sequences and aptamer technology, we have developed a new strategy to detect target molecules such as proteins. METHODS: Ovine follicle-stimulating hormone alpha subunit (oFSHalpha) was used as the model protein to generate a specific DNA aptamer via an in vitro evolutionary process. A targeted regional-mapping approach and a target-capturing assay were used to identify the binding region on the aptamer molecule. In the detection assay, referred to as "aptamer-based regionally protected PCR" (ARP-PCR), the aptamer was allowed to bind to the target protein in solution before digestion with DNase I. The region of the aptamer bound to the target was protected from DNase I cleavage. The target-binding region of the aptamer protected from the enzymatic treatment was then amplified by the PCR. RESULTS: Aptamers against oFSHalpha were generated. Six sequences of 20 selected aptamer clones were identical. This aptamer sequence was divided into 4 regions according to the aptamer's secondary structure. From examination of the target-binding ability of each region, we determined the specific binding region, for which primers were designed. With the aptamer and primers to detect oFSHalpha by means of the ARP-PCR method, we were able to detect the target protein at concentrations as low as 10(-14) mol/L. CONCLUSIONS: Combining the use of a DNA aptamer with the PCR is a potentially useful analytic tool for detection of proteins at low concentrations. .

Proteomic analyses of recombinant human follicle-stimulating hormone and urinary-derived gonadotropin preparations.

OBJECTIVE: To understand the properties of each available gonadotropin preparation, especially in terms of the differences between urinary-derived and recombinant preparations. STUDY DESIGN: Human menopausal gonadotropin (hMG), highly purified urinary-derived follicle-stimulating hormone (uFSH-HP) and recombinant FSH (rFSH) were subjected to 2-dimensional gel electrophoresis (2-DE), and protein spots were visualized by silver-staining procedures. Major spots were analyzed by mass spectrometry. Fluorescent-labeled preparations were also subjected to 2-DE to evaluate the quantities of FSH isohormones contained in each preparation. RESULTS: 2-DE and mass spectrometry analyses of hMG identified many extracellular proteins as major impurities and several plasma membrane proteins including prion proteins. Both uFSH-HP and rFSH demonstrated slight impurities and showed several alpha and beta subunit isohormones. rFSH contained higher amounts of the basic isohormones of the alpha subunit than uFSH-HP, whereas the predominance of the basic isohormones was less significant in the beta subunit. CONCLUSION: Proteomic analyses demonstrated the detailed protein profiles of each preparation. Differences in the quantities of alpha subunit isohormones may contribute to the variations in FSH activity observed between recombinant and urinary-derived FSH preparations.

Non-gonadotropin-releasing hormone-mediated transcription and secretion of large human glycoprotein hormone alpha-subunit in human embryonic kidney-293 cells.

To identify genes that are most responsive to a sustained activation of a G(s) protein-coupled receptor, HEK293 cells were stably transfected with the beta(2)-adrenergic receptor and stimulated with agonist isoproterenol (1 mum). A microarray study indicated that the gene with the highest stimulation index (500-fold) encoded the common alpha-subunit of human glycoprotein hormones (GPHalpha). Induction of GPHalpha transcription in response to cAMP elevations resulted in a dramatic increase (600-fold) of protein secretion as shown by RT-PCR and a highly specific time-resolved immunofluorometric assay. Cloning and sequencing of the GPHalpha cDNA and mass spectrometric analysis of HPLC-purified GPHalpha derived from serum-free HEK293-beta(2)-adrenergic receptor-stimulated cells verified the nature of the molecule. Enzymatic deglycosylation with subsequent Western blots revealed that this was a large hyperglycosylated form of GPHalpha that had not been associated with a beta-subunit previously. This uncombined variant is known to be either cosecreted with GPHs from the pituitary, the placenta, and a variety of tumors or secreted without GPHs from APUD cells and rare tumors. Moreover, it is similar to GPHalpha found at high concentrations in seminal plasma. As shown by a panel of endogenous or transfected G protein-coupled receptors in HEK293 cells, the expression of large GPHalpha was controlled by G(s)- and G(q)- but not G(i)-dependent receptors and mediated via cAMP and Ca(++) release. This suggests that Gq- or G(s)-coupled receptors other than the classical GnRH receptor may play a role in the regulation of nonpituitary, nonplacental GPHalpha secretion under physiological and pathological conditions.

Assembly and structural characterization of an authentic complex between human follicle stimulating hormone and a hormone-binding ectodomain of its receptor.

Follicle stimulating hormone (FSH) is secreted from the pituitary gland to regulate reproduction in vertebrates. FSH signals through a G-protein coupled receptor (FSHR) on the target cell surface. We describe here the strategy to produce a soluble FSH-FSHR complex that involves the co-secretion of a truncated FSHR ectodomain (FSHR(HB)) and a covalently linked FSHalphabeta heterodimer from baculovirus-infected insect cells. FSH binds to FSHR(HB) with a high affinity comparable to that for the full-length receptor. The crystal structure of the FSH-FSHR(HB) complex provides explanations for the high affinity and specificity of FSH interaction with FSHR, and it shows an unexpected dimerization of these complexes. Here we also compare the crystal structure with theoretical models of the FSH-FSHR-binding mode. We conclude that the FSH-FSHR(HB) structure gives an authentic representation of FSH binding to intact FSHR.

Expression and regulation of the pituitary- and placenta-specific human glycoprotein hormone alpha-subunit gene is restricted to the pituitary in transgenic mice.

Expression of the glycoprotein hormone alpha subunit occurs in both the pituitary and placenta in humans. However, this study found that expression of this subunit is restricted to the pituitary in mice. An interspecies analysis of human alpha-subunit gene regulation was undertaken, using the transgenic-mouse approach. In mice transgenic for a genomic clone containing the complete human alpha-subunit gene and several kilobases of 5'- and 3'-flanking sequences, cell-type-specific expression and hormonal regulation of the human alpha-subunit transgene occurred in the mouse pituitary, whereas no expression of the transgene was detectable in the mouse placenta. These findings provide strong evidence that a common trans-acting factor(s) regulates glycoprotein hormone alpha-subunit gene expression in the human and mouse pituitaries; however, this factor(s) or a unique factor(s), though functional in the human placenta, is either nonfunctional or absent in the mouse placenta.

Expression and purification of feline thyrotropin (fTSH): immunological detection and bioactivity of heterodimeric and yoked glycoproteins.

The goal of this study was to express and purify recombinant feline TSH as a possible immunoassay standard or pharmaceutical agent. Previously cloned feline common glycoprotein alpha (CGA) and beta subunits were ligated into the mammalian expression vector pEAK10. The feline CGA-FLAG and beta subunits were cloned separately into the pEAK10 expression vector, and transiently co-transfected into PEAK cells. Similarly, previously cloned and sequenced yoked (single chain) fTSH (yfTSH) and the CGA-FLAG sequences were ligated into the same vector, and stable cell lines selected by puromycin resistance. Expression levels of at least 1 microg/ml were achieved for both heterodimeric and yoked fTSH forms. The glycoproteins were purified in one step using anti-FLAG immunoaffinity column chromatography to high purity. The molecular weights of feline CGA-FLAG subunit, beta subunit and yfTSH were 20.4, 17, and 45 kDa, respectively. Both heterodimeric and yoked glycoproteins were recognized with approximately 40% detection by both a commercial canine TSH immunoassay and an in-house canine TSH ELISA. The yoked glycoprotein exhibited parallelism with the heterodimeric form in the in-house ELISA, supporting their possible use as immunoassay standards. In bioactivity assays, the heterodimeric and yoked forms of fTSH were 12.5 and 3.4% as potent as pituitary source bovine TSH at displacing (125)I-bTSH and 45 and 24% as potent in stimulating adenylate cyclase activity in human TSH receptor-expressing JP09 cells. However, in addition to reduced receptor binding affinity, the recombinant glycohormones produced a reduced maximal effect at maximal concentration (E(max)) suggesting the possibility of the recombinant glycohormone constructs acting as partial agonists at the human TSH receptor.

The gonadotroph origin of null cell adenomas.

OBJECTIVE: The term "null cell" adenoma was first proposed in 1980 to designate pituitary adenomas lacking clinical, biochemical and morphological markers to disclose their cell origin. DESIGN: The aim of this study was to investigate the presence of α- and ß-gonadotropin subunits in clinically nonfunctioning pituitary tumors, which were initially immunonegative and thus diagnosed as null cell adenomas. For this reason, we reapplied immunohistochemistry using a more sensitive method comprising a tyramide signal amplification technique, combined with a polymer antibody immunohistochemical detection system. RESULTS: With this approach, all these previously negative tumors became positive for α- and ß-gonadotropin hormone subunits. CONCLUSIONS: Our results prove that so-called "null cell" adenomas produce α-SU or/and ß-FSH or ß-LH and therefore are gonadotrph adenomas in origin.

Molecular and expression characterization of three gonadotropin subunits common alpha, FSHbeta and LHbeta in groupers.

A SMART cDNA plasmid library was constructed from protogyous greasy grouper (Epinephelus coioides) pituitary, and the full-length cDNAs of three gonadotropin (GTH) subunits common alpha, FSHbeta and LHbeta were cloned and sequenced from the library. The nucleotide sequences of common alpha, FSHbeta and LHbeta subunit cDNAs are 647, 594 and 574 bp in length, and encode for mature peptides of 94, 99 and 115 aa, respectively. High homology was observed by amino acid sequence alignment and identity comparison of the grouper mature peptides of common alpha, FSHbeta and LHbeta with that of other fishes. Phylogenetic tree analyses of the three GTH mature subunits revealed similar phylogeny relationships among the studied fish species. Three polyclonal antibodies were prepared from the in vitro expressed common alpha, FSHbeta and LHbeta mature proteins, respectively. Western blot analysis and immunofluoresence localization were performed on two typical stages of ovarian development stages in red-spotted grouper. Significant differences in protein expression levels of three gonadotropin subunits were revealed between the two ovarian development stages. In the individuals with resting ovary, common alpha was almost not detected in pituitaries, and FSHbeta and LHbeta expression levels were very low. While in the individuals with developing ovary, the expression of all three gonadotropin subunits reached to a high level. Immunofluoresence localization indicated that the grouper FSHbeta cells mainly distributed in the middle area of PPD, while the LHbeta cells distributed more widely, including in the area similar to the FSHbeta cells and at the external periphery of pituitary near to the PI side. The common alpha might be expressed in both FSHbeta and LHbeta cells. Double immunofluoresence localization further demonstrated FSHbeta and LHbeta expression in distinct cells in the PPD area, although the FSHbeta and LHbeta cells were detected in the identical area of PPD.

Msx1 is present in thyrotropic cells and binds to a consensus site on the glycoprotein hormone alpha-subunit promoter.

Our studies are aimed at identifying the transcription factors that activate the glycoprotein hormone alpha-subunit promoter. Therefore, we performed a Southwestern screening of a thyrotropic (alphaTSH) cDNA expression library, using the region of the promoter from -490 to -310 that contains sequences critical for expression in thyrotrope cells. A clone was isolated corresponding to part of the coding sequence of Msx1, which is a homeodomain-containing transcription factor that has been found to play an important role in the development of limb buds and craniofacial structures. Northern blot analysis, using the cloned Msx1 cDNA fragment as a probe, demonstrated that alpha-subunit-expressing thyrotrope cells (alphaTSH cells and TtT97 tumors) contained Msx1 RNA transcripts of 2.2 kb, while somatomammotrope (GH3) cells that do not produce the alpha-subunit had barely detectable levels. The presence of Msx1 protein was demonstrated by Western blot analysis in alphaTSH cells. We also demonstrated that transcripts encoding the closely related Msx2 factor were not detectable by Northern blot analysis in either thyrotrope or somatomammotrope-derived cells. Subfragments of the region from -490 to -310 of the alpha-subunit promoter were used in a Southwestern blot assay using bacterially produced Msx1 and demonstrated that binding was localized specifically to the region from -449 to -421. Deoxyribonuclease I protection analysis, using purified Msx1 homeodomain, demonstrated structurally induced differences in DNA digestion patterns between -436 and -413, and sequence analysis of this region revealed a direct repeat of the sequence GXAATTG, which is similar to the Msx1 consensus-binding site. When nucleotides at both sites were mutated, Msx1 binding was dramatically reduced, and the activity of an alpha-subunit promoter construct decreased by approximately 50% in transfected thyrotrope (alphaTSH) cells. These studies suggest that Msx1 may play a role in the expression of the alpha-subunit gene in thyrotrope cells.

The antigenic structure of the human glycoprotein hormone alpha-subunit: II. Cross-species comparisons.

Eight monoclonal antibodies, specific for the glycoprotein hormone alpha-subunit, were raised against human free alpha-subunit, human FSH, or human CG. All of these antihuman monoclonal antibodies were tested for cross-reactivity with alpha-subunits derived from bovine, porcine, equine, bull frog, sea turtle, turkey, and ostrich glycoprotein hormones. All showed cross-reactivity with affinities ranging from 10(-4) to 10(-8) depending upon the antibody and the species of alpha-subunit. Cyanogen bromide fragments of bovine and equine alpha, when tested with selected antibodies indicated that antigenic determinants could be localized in two regions: alpha 9-33 and alpha 76-92. Comparison of amino acid sequences, and relative potencies, suggest that major antigenic determinants involve residues 21, 22, and 23 (F-F-S in human alpha) and 76-85 (G-G-F-K-V-E-N-H-T-A in human alpha). As part of this study the N-terminal amino acid sequences of bull frog, sea turtle, turkey, and ostrich alpha-subunits were determined and reported for the first time.

A new substance resembling luteinizing hormone in the blood of rhesus monkeys.

Sera from a number of rhesus monkeys showed low or undetectable levels of LH according to radioimmunoassays which employ radioiodinated rhesus LH and antisera against rhesus LH or hCG. These same sera, when assayed by a system utilizing radioiodinated ovine LH and a unique anti-ovine LH serum which cross-reacts with LH from a variety of species, appeared to contain large and variable quantities of LH. The chromatographic behavior on Sephadex G-100 of the LH-like material in these sera was indistinguishable from that of authentic rhesus LH. Chromatographic fractions containing this LH-like material, as well as the sera from which they were derived, generated dose-response curves in the ovine:anti-ovine radioimmunoassay with steeper slopes than those produced by rhesus LH. These same chromatographic fractions had negligible activity in an alpha subunit radioimmunoassay which detects not only free rhesus alpha subunit but also the alpha component of undissociated rhesus glycoprotein hormones including LH. Treatment of these fractions with 4M guanidine-HCl produced a substance of smaller molecular size which, like rhLH beta, was active in the ovine:anti-ovine assay. A substance closely resembling the serum LH-like material but having a somewhat greater molecular size is also present in the rhesus adenohypophysis, but its relationship to the serum substance remains uncertain.

Carbohydrate composition of the alpha-subunit of human choriogonadotropin (hCG alpha) and the free alpha molecules produced in pregnancy: most free alpha and some combined hCG alpha molecules are fucosylated.

The carbohydrate compositions of pregnancy-derived hCG alpha (dissociated from intact hCG) and free alpha-subunit were analyzed using a combination of chemical analysis, lectin affinity chromatography, and glycosidase sensitivity. For direct compositional analysis, parallel samples were hydrolyzed in trifluoroacetic acid and analyzed for sialic acid and neutral sugars without prior derivatization. Separation of the monosaccharides was achieved by HPLC on a Dionex CarboPac column eluted at high pH, and the resolved monosaccharides were quantified by pulsed amperometric detection. The amounts of sugar that were found relative to peptide indicated the presence of two N-linked oligosaccharides per molecule on both hCG alpha and free alpha. Free alpha contained 2.5-fold higher amounts of sialic acid and galactose as well as a higher amount of N-acetylglucosamine than did hCG alpha. Free alpha also contained a 6-fold higher amount of fucose than did hCG alpha (1.2 vs. 0.2 residues of fucose/molecule). Serial fractionation of intact hCG alpha and free alpha molecules by lectin affinity chromatography indicated that virtually all of the hCG alpha-subunits contained at least one Concanavalin-A (Con-A)-binding site, whereas as many as 32% of the free alpha molecules could not bind to Con-A. Chromatography on Lens culinaris (Lch) resulted in 12% binding of hCG alpha and approximately 72% binding of free alpha (80-85% of the Con-A-bound free alpha and 47-48% of the Con-A-nonbound free alpha bound to Lch). Endoglycosidase-H (endo-H) treatment of hCG alpha released a portion of the oligosaccharides. The endo-H-released material appeared to be a monoantennary hybrid based on DEAE-binding properties and carbohydrate composition. In contrast to hCG alpha, free alpha was completely resistant to endo-H treatment. Incubation of endo-H-resistant hCG alpha with glycopeptidase-A resulted in the release of two components, which could be separated into monoantennary and biantennary fractions on the basis of size and charge. The collective data suggest that hCG alpha contains primarily monoantennary hybrid oligosaccharide structures and relatively little fucose. In contrast, free alpha contains primarily multiantennary oligosaccharide structures, and most of the free alpha molecules contain at least one oligosaccharide with fucose attached to the asparagine-linked N-acetylglucosamine residue.

The antigenic structure of the human glycoprotein hormone alpha-subunit. I. Characterization of anti-alpha monoclonal antibodies.

The glycoprotein hormones CG, LH, FSH, and TSH are composed of two noncovalently linked subunits, alpha and beta. The beta-subunit confers hormone specificity, while the alpha-subunit is homologous within a species. To help in determining the antigenic structure of the common alpha-subunit, six monoclonal antibodies (mAbs) to the free or heterodimeric alpha-subunit of human (h) gonadotropic hormones have been prepared and, along with two previously isolated mAbs, have been characterized for binding specificity to alpha- and beta-subunits and the human glycoprotein hormones, CG, LH, FSH, and TSH. Each mAb was derived from hybidomas of FO myeloma cells fused with spleen cells from mice immunized with free alpha-subunit, hCG or hFSH. mAbs A101, A102, and E512 were specific for the alpha-subunit but showed the highest affinity for the intact hormone; K2.18, K94.6, E501, E502, and E511 were specific for free alpha. All of the antibodies inhibited binding of 125I-hCG to luteal membrane receptor, and 125I-labeled mAbs did not recognize hCG/receptor complex. Characterization by two-site binding assays using alpha, hCG, or hFSH as antigen revealed that all the mAbs bind to unique sites on alpha which may be overlapping, and which are modified in the intact hormone. The antigenic sites for mAbs E502, E511, and K2.18 are at least partially linear because they bind to reduced, carboxymethylated alpha.