Differences in the carbohydrate moieties of the common alpha-subunits of human chorionic gonadotropin, luteinizing hormone, follicle-stimulating hormone, and thyrotropin: preliminary structural inferences from direct methylation analysis

The carbohydrate components of combined alpha-subunits of urinary hCG and human pituitary LH (hLH), FSH (hFSH), and TSH (hTSH), each derived from the intact hormone, were studied by direct sugar analysis and methylation analysis. The methods provide a complete survey of the structural elements contained in the complex sugars associated with these glycoproteins, but do not establish the sugar sequences or anomeric configurations of glycosidic bonds. By analogy to N-linked oligosaccharides that occur in many glycoproteins, the data suggest distinct structural features for carbohydrates of alpha-subunits combined with beta-subunits. hCG alpha contains biantennary asparagine-linked chains terminated by either NeuAc alpha 2-3Gal beta 1- or GlcNAc beta 1-2 Man alpha 1- and lacks fucose. hTSH alpha contains biantennary chains with the same termini as hCG alpha plus terminal R-O-4GalNAc and a fucosyl residue linked alpha 1-6 to the inner GlcNAc residue of the N-linked chitobiosyl core. hLH alpha may contain some high mannose chains, but primarily contains biantennary chains terminated by NeuAc alpha 2-3(6)Gal beta 1-, GlcNAc beta 1-, GalNac-1-, R'-O-6GlcNAc-1-, and R"-0-2Man-1-plus a fucosyl residue linked alpha 1-6 to the inner GlcNAc residue of the N-linked chitobiosyl core. hFSH alpha contains more complicated structures that probably include a bisecting GlcNAc residue linked beta 1-4 to a 3,6-di-O-substituted core mannosyl residue, and terminal NeuAc alpha 2-3Gal beta 1-4(+/- Fuc alpha 1-3)GlcNAc-1, Gal beta 1-4(+/- Fuc alpha 1-3)GlcNAc-1-, R"'-O-GalNAc-1-, and GalNAc-1. In addition, the presence of 2,4-di-O-substituted mannose in hFSH alpha indicates that it contains triantennary chains. The identities of the R; R', R", and R"' groups were not determined, but recent studies of glycoprotein hormones suggest that they may be sulfate groups. Our results demonstrate differential glycosylation of virtually identical polypeptide hormone alpha-subunits produced in the same organ or perhaps even in the same cell.     

Beta-core fragments are contaminants of the World Health Organization Reference Preparations of human choriogonadotrophin and its alpha-subunit

Highly purified preparations of human choriogonadotrophin (hCG), hCG alpha and hCG beta, including those preparations which are being distributed by the World Health Organization as International Standards, cross-reacted in a new radioimmunoassay with increased relative specificity for the beta-core fragment of hCG. A major portion of the beta-core immunoreactivity in the hCG and hCG alpha preparations eluted from Sephadex G-100 in a position (approximate apparent molecular size 15,000-18,000) corresponding to that of purified beta-core fragment prepared from pregnancy urine. However, in the case of hCG beta-subunit preparations, virtually all of the beta-core cross-reacting material eluted from Sephadex G-100 in the same fractions as the native hCG beta-subunit. Quantitatively, the cross-reacting beta-core material accounts for less than 1% (w/w) of the total hCG or subunit immunoreactivity, as measured by conventional radioimmunoassays. The presence of the beta-core fragments as discrete molecular components of the hCG and hCG alpha preparations should be borne in mind when these preparations are used to calibrate new radioimmunoassays for hCG-related molecules.     

The O-linked oligosaccharide structures are striking different on pregnancy and choriocarcinoma HCG

hCG is a glycoprotein hormone which is detected in the serum and urine of pregnant women and of patients with hydatidiform mole and choriocarcinoma. The molecule contains 4 O-linked sugar chains. In an effort to identify cancer markers, the structures of these sugar units on the hCG produced in pregnancy and choriocarcinoma were compared. hCG molecules in patient urines were purified by immuno-affinity chromatography and gel filtration. beta-elimination was used to cleave the O-linked sugar units, radioactive sodium borohydride to label them, and gel filtration on Bio-Gel P4 to size them and compare their elution volumes with those of standard oligosaccharides of known structure. A trisaccharide, NeuAc alpha 2-3Gal beta 1-3GalNAc-, was found to be the principal unit attached to urinary hCG from pregnant women (10 samples). A hexasaccharide, NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-3Gal beta 1-4 GlcNAc beta 1-6)GalNAc-, which accounted for just 6% (mean, range 0-14%) of the O-linked sugar units on pregnancy hCG, was the principal unit (mean 52% of total, range 50-56%) attached to the hCG from choriocarcinoma patient urines (3 samples). These results indicate that hexasaccharide-abundant hCG is an indicator of choriocarcinoma.     

Distribution of O-linked sugar units on hCG and its free alpha-subunit

hCG, a glycoprotein hormone produced by the trophoblast in pregnancy, is composed of two dissimilar subunits, alpha and beta, joined non-covalently. hCG has four O-linked sugar units, all attached to the beta-subunit. The trophoblast also produces a free form of the alpha-subunit, which unlike the alpha-component of hCG, can contain an O-linked sugar unit. The structures of the O-linked sugar units were examined. Four structures were identified on urinary hCG. A hexasaccharide, NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-6)GalNAc- accounting for 13%, a tetrasaccharide, NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-6)GalNAc-, for 34%, a trisaccharide, NeuAc alpha 2-3Gal beta 1-3GalNAc-, for 43% and a disaccharide, NeuAc alpha 2-6GalNAc- for 10% of the total O-linked sugar structures. Similar mixtures were found on peptides containing one, three or four sugar units suggesting a random distribution among attachment sites. The distribution of O-linked sugar structures on hCG and free alpha-subunit from trophoblast explant cultures was compared. The mixture of structures attached at the single site on the free alpha-subunit paralleled that at the four sites on the hCG.     

Recognition by the glycoprotein hormone-specific N-acetylgalactosaminetransferase is independent of hormone native conformation.

Some members of the glycoprotein hormone family [luteinizing hormone (LH), thyroid-stimulating hormone (TSH), and free alpha subunit] bear unique asparagine-linked oligosaccharides with the terminal sequence SO4-Gal-NAc beta 1,4GlcNAc beta 1,2Man alpha, whereas other members [human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH)] bear predominantly oligosaccharides terminating in the sequence sialic acid alpha-Gal beta 1,4GlcNAc beta 1,2Man alpha. We previously identified an N-acetylgalactosaminetransferase (GalNAc-transferase) in bovine pituitary membranes that specifically recognizes the alpha subunit peptide and adds GalNAc to the synthetic intermediate GlcNAc2Man3GlcNAc2. In the current study we demonstrate that bLH, hCG, hCG beta, hCG alpha, and FSH alpha are recognized by the pituitary GalNAc-transferase in vitro, whereas oFSH, hFSH, and hFSH beta are not (b-, h-, and o-indicate bovine, human, and ovine). The apparent Km values for addition of GalNAc to oligosaccharides on hCG alpha and hCG beta, 13.0 and 6.2 microM, respectively, are not altered by reduction and alkylation. Thus, recognition of the peptide determinant does not require maintenance of native tertiary structural features. In the presence of the recognition determinant the Km for addition of GalNAc to the intermediate GlcNAc2Man3GlcNAc2 is reduced from 1.2-2.6 mM to less than 13 microM. hFSH is not efficiently recognized by the GalNAc-transferase due to the absence of the recognition marker on hFSH beta and some degree of masking of the recognition marker on the alpha subunit when combined with FSH beta. Since recognition is directed at primary and possibly secondary structural features, it should be possible to determine which regions of the alpha and beta subunits are responsible for the specificity of the GalNAc-transferase.     

The origin of a human chorionic gonadotropin beta-subunit-core fragment excreted in the urine of patients with cancer

Immunoreactive human Chorionic Gonadotropin (hCG), its subunits and hCG beta-core fragment were analyzed, using Sephadex G-100 chromatography, in urine and tumour extracts from four patients with cancer. These patients were selected for investigation because they were excreting proportionally large amounts of the hCG beta-core fragment in their urine. Although 30-85% of the total immunoreactive urinary hCG was hCG beta fragment, traces of the fragment (2% of total hCG) were found in only two of the tumours and none in the other two. The predominant molecular form of hCG in the tumours was intact free beta-subunit of hCG. The conclusion is that the hCG beta-core fragment found in the urine of some patients with cancer is not a secretion product of the tumours. This fragment is very likely a peripheral degradation product of the free beta-subunit of hCG which is secreted by the tumours.     

Carbohydrate composition of beta-core

beta-Core is a major component of the hCG-related molecules found in pregnancy urine. We previously have purified the beta-core molecule and have deduced portions of its carbohydrate structure based on lectin binding data. In the present study we used recently developed technology to determine the carbohydrate composition of beta-core and hCG beta (CR119). 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 beta-core and hCG beta. hCG beta contained additional sugars consistent with the presence of four O-linked oligosaccharides. Compared to hCG beta, beta-core contained negligible sialic acid, galactose, or N-acetylgalactosamine. The compositional data suggest that beta-core does not contain N-acetylglucosamine at the nonreducing end of the molecule, whereas the trimannosyl-chitobiose core is apparently intact at both glycosylation sites, consistent with the ability of the molecule to bind to Concanavalin-A. Comparable fucose contents and abilities of beta-core and hCG beta to bind to Lens culinaris indicate a similar extent of fucosylation on the internal N-acetylglucosamine in both molecules. We propose that the N-linked oligosaccharides on beta-core closely resemble the underlying N-linked structures of hCG beta with the antennary sialic acid, galactose, and N-acetylglucosamine removed.     

Renal metabolism of the beta-subunit of human choriogonadotropin in the rat

We analyzed the immunoreactive renal metabolites of the beta-subunit moieties of unlabeled highly purified hCG, hCG beta, and desialylated hCG (as-hCG) in rats by RIA and Sephadex G-100 chromatography. Infusions of hCG beta, as-hCG, or intact hCG resulted in accumulation in the kidney of a large quantity of small mol wt peptides lacking the immunological determinants of the carboxy-terminal peptide (CTP) of the beta-subunit. In the case of as-hCG, renal accumulation of these beta-core fragments was greatly enhanced when as-hCG binding to hepatic galactose receptors was inhibited by infusion of as-fetuin. The beta-core fragments in kidney had the same immunological and G-100 chromatographic characteristics as beta-core fragments in liver, suggesting similar intracellular catabolic mechanisms in these tissues. The kinetics of beta-core fragment turnover in kidney were studied after injection of hCG beta, which is cleared from the circulation within 1 h. Loss of beta CTP immunoreactivity was the initial event in hCB beta catabolism by the kidney; most of this process occurred between 7 and 30 min after injection. This was followed by a gradual reduction of the size of accumulated hCG beta metabolites over the next 60 min. The beta-core fragments that accumulated had a Kav of approximately 0.57 and a very slow degradation rate over the next 5 h (half-life greater than 6 h). Chromatographic analysis of urine obtained 6 h after beginning a continuous infusion of hCG, hCG beta, or as-hCG displayed in each case a major peak corresponding to the infused molecule, apparently intact, and a minor peak of beta CTP immunoreactivity of small mol wt. Relative to the beta CTP fragments apparent in urine, there were few beta-core fragments. These data indicate that separate fates exist for immunoreactive fragments generated by hCG beta metabolism in the rat kidney. One appears to be intracellular and similar to the liver pathway of as-hCG degradation in that it leads to the formation of long-lived beta-core fragments. The other takes place within ready access to the urinary compartment and leads to the accumulation in urine of beta-CTP fragments.     

Structural requirements for sulfation of asparagine-linked oligosaccharides of lutropin.

Human and bovine pituitary glycoprotein hormones (lutropin, follitropin, and thyrotropin) contain varying amounts of N-acetylgalactosamine and sulfate. The sulfate on asparagine-linked oligosaccharides of bovine lutropin (bLH) is present exclusively on GalNAc in the sequence GalNAc(beta 1-4)GlcNAc(beta 1-2)Man alpha. We have examined the structural requirements for sulfation of bLH oligosaccharides by using a reconstituted cell-free system. After cleavage from the protein, oligosaccharides containing the sequence GalNAc(beta 1-4)Glc-NAc(beta 1-2)Man alpha were sulfated by enzymes in pituitary membranes. Addition of one or two sulfates was observed, depending upon the number of GalNAc acceptor sites on the oligosaccharide. Neither GalNAc alone nor oligosaccharides devoid of GalNAc were sulfated. Membranes from placenta or liver did not sulfate oligosaccharides released from bLH, indicating that the sulfating activity is pituitary-specific. The lack of peptide dependence for sulfation, in conjunction with the oligosaccharide specificity, suggests that the sequence GalNAc(beta 1-4)GlcNAc(beta 1-2)Man alpha contains the recognition signal for the sulfotransferase(s).     

Beta-core fragment is a major form of immunoreactive urinary chorionic gonadotropin in human pregnancy

A small mol wt fragment of the beta-subunit of hCG (beta-core fragment) is present in the urine, but not the serum, of pregnant women. We evaluated the relative proportions of this immunoreactive, but biologically inactive, fragment in urine from 15 women at different stages of pregnancy. Freshly voided urine was ultrafiltered and concentrated, and the molecular species of immunoreactive hCG were separated by Sephadex G-100 column chromatography. All urine samples contained the beta-core fragment, which eluted after the alpha-subunit of hCG. This fragment lacked the carboxy-terminal epitope of hCG, was inactive as an in vitro bioassay, and adsorbed to Concanavalin-A. The beta-core fragment was a major form the immunoreactive hCG in urine throughout pregnancy and accounted for over 90% of the immunoreactive hCG in urine from midpregnancy. The excretion pattern of the beta-core fragment can account for the low biological to immunological ratio of urinary hCG that occurs at different stages of pregnancy.     

Molecular basis of recognition by the glycoprotein hormone-specific N-acetylgalactosamine-transferase.

Lutropin (LH) bears asparagine-linked oligosaccharides terminating with the unique sequence SO4-4GalNAc beta 1-4GlcNAc beta 1-2Man alpha, whereas follitropin (FSH) bears oligosaccharides terminating predominantly with the sequence Sia alpha-Gal beta 1-4GlcNAc beta 1-2Man alpha, where Sia is sialic acid. We previously identified a glycoprotein-hormone-specific N-acetylgalactosamine-transferase (GalNAc-transferase) that recognizes a peptide-recognition marker(s) present on the common glycoprotein hormone alpha subunit and beta subunits of human chorionic gonadotropin and LH but not on the beta subunit of FSH. We have now identified an amino acid sequence motif, Pro-Leu-Arg, that is essential for recognition by the GalNAc-transferase. This tripeptide sequence is found 6-9 residues on the amino-terminal side of a glycosylated asparagine on the alpha subunit and beta subunits of LH and human chorionic gonadotropin but is not present on the beta subunit of FSH. The presence of this motif accounts for the differences in LH and FSH oligosaccharide structures. Additional proteins containing this recognition motif have been identified and were determined to bear sulfated oligosaccharides with the same structures as those on the glycoprotein hormones, indicating that these structures are not restricted to the glycoprotein hormones.     

Pathway of protein glycosylation in the trypanosomatid Crithidia fasciculata.

Cells of the insect parasite Crithidia fasciculata incubated with [14C]glucose were found to possess only one lipid-bound oligosaccharide with solubility in chloroform/methanol/water mixtures and net charge similar to the charges of dolichol pyrophosphate derivatives. The saccharide moiety could be released from lipid by mild acid hydrolysis. Several enzymatic and chemical treatments of the oligosaccharide indicated that the latter had the structure Man alpha leads to Man alpha leads to Man alpha leads to [Man alpha leads to Man alpha leads to Man (alpha 1 leads to 6)]Man leads to GlcNAc(beta 1 leads to 4)GlcNAc. Two labeled oligosaccharides were liberated from proteins by a sequential treatment with a protease and endo-beta-N-acetylglucosamindase H. One of the protein-bound oligosaccharides had the same structure as the lipid-linked compound, whereas in the second oligosaccharide some mannose residues had been replaced by galactose units, but both compounds migrated as did a Man7GlcNAc standard. These were the largest oligosaccharides obtained even after short labeling periods. It is suggested that glycosylation of proteins in the protozoan Crithidia fasciculata does not involved glucosylated lipid-bound oligosaccharides as intermediates.     

Subunit-specific functions of N-linked oligosaccharides in human thyrotropin: role of terminal residues of alpha- and beta-subunit oligosaccharides in metabolic clearance and bioactivity.

The recombinant human thyroid stimulating hormone (rhTSH) containing oligosaccharides terminated with NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc beta 1 showed higher in vivo activity and lower metabolic clearance rate (MCR) than pituitary human TSH (phTSH), which contains oligosaccharides terminating predominantly in SO(4)4GalNAc(beta 1-4)GlcNAc beta 1. To elucidate the relative contribution of the sulfated and sialylated carbohydrate chains of each subunit in the MCR and bioactivity of the hormone, the alpha and beta subunits of phTSH, rhTSH, and enzymatically desialylated rhTSH (asialo-rhTSH; asrhTSH) were isolated, their oligosaccharides were analyzed, and the respective subunits were dimerized in various combinations. The hybrids containing alpha subunit from phTSH or asrhTSH showed higher in vitro activity than those with alpha subunit from rhTSH, indicating that sialylation of alpha but not beta subunit attenuates the intrinsic activity of TSH. In contrast, hybrids with beta subunit from rhTSH displayed lower MCR compared to those with beta subunit from phTSH. The phTSH alpha-rhTSH beta hybrid had the highest in vivo bioactivity followed by rhTSH alpha-rhTSH beta, rhTSH alpha-phTSH beta, phTSH alpha-phTSH beta, and asrhTSH dimers. These differences indicated that hybrids with beta subunit from rhTSH displayed the highest in vivo activity and relatively low MCR, probably due to higher sialylation, more multiantennary structure, and/or the unique location of the beta-subunit oligosaccharide chain in the molecule. Thus, the N-linked oligosaccharides of the beta subunit of glycoprotein hormones have a more pronounced role than those from the alpha subunit in the metabolic clearance and thereby in the in vivo bioactivity. In contrast, the terminal residues of alpha-subunit oligosaccharides have a major impact on TSH intrinsic potency.     

Effect of individual N-glycosyl chains in the beta-subunit on the conformation of human choriogonadotropin

Human choriogonadotropin is a heterodimeric glycoprotein hormone comprised of noncovalently associated alpha- and beta-subunits. Each of the subunits has two N-glycosyl chains. In our previous communication, we investigated the role of individual carbohydrate chains in the alpha-subunit on the signal transduction function and conformation of the hormone. This paper deals with the effect of individual or both N-glycosyl chains in the beta-subunit on the function and conformation of the monomer as well as of the heterodimer. Three mutants each of hCGbeta and hCG lacking N-glycosyl chains at beta13Asn, beta30Asn and beta13,30Asn were prepared by site-directed mutagenesis by replacing Thr residues in the recognition triplet sequence at beta15 and beta32 positions with Gln. All mutant heterodimers had receptor binding and cAMP and progesterone stimulating activities comparable to wild type hCG. While the loss of carbohydrate at beta13Asn or beta13,30Asn in the case of hCGbeta monomer resulted in a 4-6%, decrease in the ordered structure, the loss of the glycosyl chain at beta 30Asn did not alter the conformation as compared with the wild type hCGbeta. Similarly, all carbohydrate deficient hCG heterodimers had a decrease of 6-8%) in the ordered structure as compared with hCG. Thus, while the individual N-glycosyl chains did not affect the function of the hormone, they did have marked effect on its conformation but the conformational changes were localized and did not perturb the receptor binding and signal transduction sites.     

Immunoreactive beta-core-like material in normal postmenopausal urine: human chorionic gonadotrophin or LH origin? Evidence for the existence of LH core.

Material with the immunochemical properties of the beta-core of human chorionic gonadotrophin (hCG) can be found in the urine of normal postmenopausal women. However, we have been unable to detect intact hCG (using an assay which is specific for the alpha-beta heterodimer of intact hCG) in serum of such subjects. The levels of serum LH and urinary beta-core were compared in matched samples from 28 women (serum LH: median 27 U/l, range 4-70 U/l, urinary beta-core: median 0.27 microgram/l, range less than 0.05-0.645 microgram/l). Urine (4 litres) from three postmenopausal women was concentrated, dialysed and subjected to gel exclusion chromatography on Sephadex G-100. Fractions were analysed by specific assays for LH, intact hCG, total beta-hCG (free beta-subunit and intact hCG), free alpha-subunit and beta-core. Material eluting at the expected position of the beta-core fragment of hCG was detected in all three samples by the beta-core, beta-hCG and LH assays, despite the fact that the LH antibody does not recognize the authentic beta-core of pregnancy. Electrophoresis and Western blotting of the concentrated urines revealed that material of the same molecular size as beta-core was recognized by the antibody to LH but not by a monoclonal antibody raised to free beta-hCG which also recognizes the beta-core molecule of hCG. We conclude that the predominant core-like material identified in postmenopausal urine is probably derived from the beta-subunit of LH.     

Structure of the human chorionic gonadotropin beta-subunit fragment from pregnancy urine

A major portion of the hCG immunoreactivity detectable in pregnancy urine is derived from a fragment of hCG beta. This lacks the COOH-terminal portion of hCG beta, but retains immunoreactivity with most antibodies raised against the beta-subunit of hCG. To improve clinical measurements of hCG and assess the importance of such fragments in human urine, we have isolated and determined the structure of this molecule. The hCG beta fragment was isolated from a partially purified commercial preparation of hCG (Organon) by gel filtration and immunoaffinity chromatography using monoclonal antibodies. It was found to consist of two polypeptide chains composed of residues beta-(6-40) disulfide-bridged to residues beta-(55-92). It also differs from the beta-subunit of hCG in its carbohydrate structure, lacking sialic acid and having a low but variable amount of galactose. A beta-fragment containing the same two NH2-terminal sequences was also isolated from a single pregnant woman's urine. The two major polypeptides comprising the beta-fragment contain a total of nine half-cystine residues, raising the possibility that a free thiol may exist or that a third undetected disulfide-bridged peptide is present in the intact fragment. However, tests for the presence of a free thiol have been negative. Another intrinsic characteristic of the beta-fragment is the formation of a variable amount of dimer in solutions of neutral pH. beta-fragment will not combine with intact alpha-subunit. Despite the absence of regions beta-(1-5), beta-(41-54), and beta-(93-145), the beta fragment is recognized by the SB-6 antibody and most monoclonal antibodies elicited to the beta-subunit, thus excluding half of the amino acids of the beta-subunit from the epitope(s) where these antibodies bind.     

Separation of positional isomers of oligosaccharides and glycopeptides by high-performance anion-exchange chromatography with pulsed amperometric detection.

High-performance anion-exchange (HPAE) chromatography under alkaline conditions (pH congruent to 13) has been found to efficiently separate neutral oligosaccharides (triose to undecaose) according to molecular size, sugar composition, and linkage of monosaccharide units. The method was able to resolve 1----3, 1----4, and 1----6 positional isomers of neutral oligosaccharides, which are defined as having the same number, type, sequence, and anomeric configurations of monosaccharides but differing in the linkage position of a single sugar. From correlating structural features of different oligosaccharides and retention times, we deduced that at least two factors are operative to determine the superior resolution of oligosaccharides by this type of chromatography: (i) the relative acidities of the hydroxyl groups and (ii) the accessibility of oxyanions of the oligosaccharides to the functional groups of the stationary phase. Splitting of peaks attributable to mutarotation was not observed. Reducing oligosaccharides were much more retained than their reduced counterparts. Linkage of Fuc(alpha 1-3) to GlcNAc of oligosaccharides markedly decreased retention times. Positional isomers of two branched monosaccharides, which differed by 1----6 and 1----4 linkages, were widely separated. The separation of 1----3 and 1----4 positional isomers of both tetrasaccharides and glycopeptides containing undecasaccharides demonstrated the significant improvement in resolution of HPAE compared to previous chromatographic methods by either reverse-phase or amine-bonded stationary phases. Picomole quantities of underivatized oligosaccharides have been detected by triple-pulse amperometric detection, which produced similar responses for a wide range of structures. Quantification of two triantennary glycopeptides from bovine fetuin by using either detector response or 1H NMR was comparable. The N-glycanase-catalyzed release of two 1----4 and 1----3 positional isomers of an undecasaccharide from a tryptic glycopeptide of bovine fetuin could be observed and quantified by direct injection of the enzyme mixture into the chromatograph.     

An enzyme releasing lacto-N-biose from oligosaccharides.

alpha-L-Fucosidase (alpha-L-fucoside fucohydrolase; EC 3.2.1.51) preparations from Streptomyces sp. 142 were found to contain an enzyme specific for lacto-N-biosidic (Gal beta 1-3GlcNAc beta 1-) linkages (type 1 structure) in oligosaccharides. The enzyme preparation, which was eluted after alpha-fucosidase from a CM-Sepharose column, contained some alpha-fucosidase activity but was free from other glycosidases and proteases. Substrate specificity studies with oligosaccharides labeled with 2-aminopyridine showed that the enzyme specifically hydrolyzed lacto-N-tetraose (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc) but did not hydrolyze lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc), lacto-N-triose, sialyl lacto-N-tetraose, lacto-N-fucopentaose I, II, or III, asialo-GM1 tetrasaccharide, or poly-N-acetyllactosamine. Structural analysis of the enzyme digest of the N-acetyllactosamine type of triantennary sugar chain with type 1 structure showed that lacto-N-biose (Gal beta 1-3GlcNAc) and the N-acetyllactosamine type of biantennary sugar chain were produced. Thus this enzyme was tentatively named lacto-N-biosidase, because it hydrolyzes oligosaccharides containing a type 1 structure at the nonreducing terminus and produces lacto-N-biose.     

Structural and functional studies of the tryptic core of the human chorionic gonadotropin beta-subunit

The beta-subunit of hCG was digested with trypsin to produce a modified form of the subunit for structure-function and immunological studies. After digestion of hCG beta with trypsin, the residual disulfide-linked core was isolated and found to be lacking the carboxy-terminal peptide (residues 115-145) and to contain bond cleavages between residues 2-3, 43-44, 74-75, and 95-96. The locations of these bond cleavages within the disulfide-bridged core were identified by isolation of the following peptides after reduction and S-carboxymethylation of the trypsin beta-core: beta 1-43, beta 3-43, beta 44-74, beta 44-95, beta 75-95, and beta 96-114. The circular dichroic spectrum of the tryptic beta-core over the wavelength region of about 200-320 nm was similar to that of the native subunit. In addition, the tryptic beta-core retained nearly full immunopotency in both polyclonal and monoclonal competitive RIAs and could combine with complementary native alpha-subunit. The hybrid, composed of the tryptic beta-core and native alpha, was purified and displayed a molar potency of about 0.1% relative to intact hCG in both a radioreceptor assay and an adenylate cyclase assay. Thus, the hybrid retained little biological activity. Although the extensive bond cleavages in the tryptic beta-core did not appear to change its secondary and tertiary structure sufficiently to significantly alter the circular dichroic spectrum, the immunoreactivity, or the capability to combine with its alpha-subunit complement, the biological functional integrity of the tryptic beta-core-containing hybrid was essentially abolished. Hence, the tryptic beta-core provides a useful derivative for detailed structure-function studies aimed at defining the necessary determinants for subunit association, receptor binding, and subsequent biological actions.     

Association of N-glycosylation of apolipoprotein B-100 with plasma cholesterol levels in Watanabe heritable hyperlipidemic rabbits.

We have previously demonstrated the heterogeneity of N-linked sugar chains of apolipoprotein (apo) B-100 in Watanabe heritable hyperlipidemic (WHHL) rabbit and fasting Japanese White rabbits (Arteriosclerosis, 10 (1990) 386-393). To investigate further the role of N-linked sugar chains of apo B-100 in lipid metabolism, we examined the correlation between the N-glycosylation of apo B-100 and serum cholesterol levels in WHHL rabbits. The N-linked sugar chains of apo B-100 were liberated by hydrazinolysis, followed by NaB3H4 reduction and were fractionated by paper electrophoresis and BioGel P-4 column chromatography. These were found to consist of one neutral (N) and two acidic fractions (A1 and A2). N contained a high mannose type oligosaccharide consisting of Man5.GlcNAc2 to Man9.GlcNAc2, while A1 and A2 contained monosialylated and disialylated complex type oligosaccharides, respectively. The molar ratio varied among the 5 WHHL rabbits. There was an inverse correlation between the ratio of acidic oligosaccharide fractions (A1 + A2) and serum cholesterol levels (r = -0.971, P less than 0.01) in the 5 WHHL rabbits. These results indicate that the N-glycosylation of apo B-100 is closely related to cholesterol metabolism in WHHL rabbits.