The lipid transport system in the mouse, Mus musculus: isolation and characterization of apolipoproteins B, A-I, A-II, and C-III

Four of the principle apolipoproteins of murine serum have been isolated and characterized. On the basis of their physicochemical properties, they are homologous with the human and rat apoA-I, A-II, B, and C-III. The group of apolipoproteins of middle to low molecular weight, i.e., A-I, A-II and C-III, were separated from the protein moiety of high density lipoproteins (HDL) by gel filtration chromatography, followed by electrophoresis in alkaline-urea polyacrylamide gel with electrophoretic elution. Murine apoA-I, the major protein of HDL (60-80%) displayed an Mr of approximately 27,000, and was polymorphic (four prominent isoproteins with isoelectric points in the range of pH 5.5-5.7). The amino acid profiles of mouse, rat, and human apoA-I generally resembled each other, the former being distinguished by a content of one isoleucine residue per mole. Amino terminal sequence analysis revealed marked homology between the mouse, rat, dog, and human proteins; mouse and rat apoA-I differed at residues 9 and 18 with potential dissimilarities at residues 5 and 15, while the murine and canine sequences were distinct at residues 6, 9, 13, 15, and 30. Apolipoprotein A-II was a monomer, exhibiting an Mr approximately 11,000 in SDS gels; in addition, it was polymorphic (three apparent isoproteins with pI in the pH range 5.05-5.2), and resembled its human and rat counterparts in amino acid composition. ApoC-III, an acidic peptide of pI 4.74 and of Mr approximately 9,600, possessed an amino acid composition very like that of the homologous human and rat proteins. The homology of mouse apoC-III with the human protein was confirmed by NH2-terminal sequence analysis, which revealed identical amino acids in six positions (1, 2, 4, 8, 9, and 13). As shown earlier (Camus et al. 1983. J. Lipid Res. 24: 1210-1228), two forms of immunologically reacting apoB predominated in mouse VLDL and LDL. After isolation of these lipoproteins in the presence of 1 mM PMSF, the apparent sizes of the high and low Mr forms, apoBH and apoBL, were in the ranges approximately 400,000-530,000 and approximately 250,000-280,000, respectively, according to the SDS gel system. We observed that inclusion of 1 mM PMSF was essential to retard degradation of the high Mr form apoBH. The murine B proteins were isolated from apoVLDL and apoLDL by gel filtration chromatography on Sephadex G150 in anionic detergent, and displayed apparent Mr values of 460,000 (apoBH) and 250,000 (apoBL) in 3% SDS gels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular cloning of a human apoC-III variant: Thr 74----Ala 74 mutation prevents O-glycosylation

Apolipoprotein C-III (apoC-III) is a major protein of very low density lipoprotein (VLDL). The apoC-III polypeptide contains a carbohydrate chain containing galactosamine, galactose, and sialic acid attached in O-linkage to a threonine residue at position 74. We have cloned the apoC-III gene from a subject whose serum contained unusually high amounts of apoC-III lacking the carbohydrate moiety (C-III-0). DNA sequence analysis of the cloned gene revealed a single nucleotide substitution (A----G) that encodes an alanine at position 74 instead of the normal threonine. As a result of this amino acid replacement, the mutant apoC-III polypeptide is not glycosylated. The mutation in the apoC-III gene creates a novel AluI site that permits diagnosis of the change by Southern blotting of genomic DNA.     

Characterization of the oligosaccharide side chain of apolipoprotein C-III from human plasma very low density lipoproteins.

Apolipoprotein C-III1 and apolipoprotein C-III2 each contain one oligosaccharide side chain, bound O-glycosidically to threonine in position 74 of the amino acid sequence. The studies reported in this paper characterize these alkali labile oligosaccharides, thereby demonstrating the complete structure of apolipoprotein C-III. Monosaccharide analysis revealed the following sugar composition: D-galactose/N-acetyl-D-galactosamine/sialic acid 1 : 1 : 1 and 1 : 1 : 2 for apolipoprotein C-III1 and apolipoprotein C-III2, respectively. Treatment of desialylated apolipoproteins with alkaline borohydride released the reduced disaccharide beta-D-galactosyl-(1 leads to 3)-N-acetyl-D-galactosaminitol, which was detected by gas-liquid chromatography. Further studies employing periodate oxidation and Smith degradation indicated that the structure of the trisaccharide from apolipoprotein C-III1 was alpha-N-acetylneuraminyl-(2 leads to 3)-beta-D-galactosyl-(1 leads to 3)-N-acetyl-D-galactosaminitol. The tetrasaccharide structure from apolipoprotein C-III2 is made up of this trisaccharide plus one sialic acid residue linked to C6 of N-acetyl-D-galactosaminitol, as was shown by the assessment of chromogens formed upon alkaline degradation.     

Amino acid sequence of human plasma apolipoprotein C-III from normolipidemic subjects

The complete amino acid sequence of human plasma apolipoprotein C-III (apoC-III) isolated from normal subjects is described. ApoC-III is a linear polypeptide chain of 79 amino acids. Tryptic digestion of intact apoC-III produced 5 major peptides, while tryptic digestion of the citraconylated protein yielded two peptides. The complete amino acid sequence of apoC-III was determined by the automated Edman degradation of the intact protein as well as the various tryptic peptides. Phenylthiohydantoin amino acids were identified by high-performance liquid chromatography and chemical ionization mass spectrometry. The amino acid sequence of apoC-III isolated from normolipidemic subjects is identical to the apoC-III sequence derived from the cDNA sequence and differs at 4 positions from the previously reported sequence of apoC-III derived from a patient with type V hyperlipoproteinemia.     

Isolation and characterization of cDNA clones corresponding to two different human apoC-III alleles

We have recently reported that the human apolipoprotein A-I (apoA-I) and apolipoprotein C-III (apoC-III) genes are physically linked and that the presence of a DNA insertion in the apoA-I gene is correlated with apoA-I-apoC-III deficiency in patients with premature atherosclerosis. In addition, the presence of a polymorphic restriction endonuclease site (SacI) in the 3' noncoding region of apoC-III mRNA has been correlated with hypertriglyceridemia in humans. In this study, we report the isolation and characterization of cDNA clones containing the entire apoC-III mRNA coding sequence. The nucleotide-derived apoC-III amino acid sequence indicates that the apoC-III primary translational product contains a 20 amino acid N-terminal extension, which conforms with the general properties of known signal peptides, and is highly homologous to the recently reported rat apoC-III signal peptide. The DNA-derived apoC-III amino acid sequence differs from the previously reported apoC-III amino acid sequence at four amino acid residues. More specifically, at positions +32, +33, +37, +39, the DNA sequence predicts Glu, Ser, Gln, Ala, respectively, while the previously reported sequence specifies Ser, Gln, Ala, Gln, respectively. Finally, isolation and characterization of apoC-III cDNA clones, with or without the polymorphic SacI restriction site, indicated that the apoC-III nucleotide sequence corresponding to the Sac+ and Sac- clones differs at three nucleotide sites; however, the amino acid sequence specified by the Sac+ and Sac- alleles is identical.     

Three genetic variants of human plasma apolipoprotein A-IV. apoA-IV-1(Thr347----Ser), apoA-IV-0(Lys167----Glu,Gln360----His), and apoA-IV-3(Glu165----Lys).

Human apolipoprotein (apo) A-IV is a genetically polymorphic glyco-protein of 376 residues. We have recently reported the molecular basis for the two most common isoproteins, apoA-IV-1 and apoA-IV-2(Gln360----His), and for two rare variants, apoA-IV-0 (4-amino acid insertion) and apoA-IV-3(Glu230----Lys). In this report, we present the genetic basis for three additional isoproteins of apoA-IV. Sequence analysis of the apoA-IV-1 allele revealed a common nucleotide substitution which converts threonine (ACT), residue 347 of the mature protein, into serine (TCT). In one out of the five subjects with the apoA-IV-1/0 phenotype we identified two point mutations: 1) replacing the positively charged lysine (AAG), amino acid 167, with a negatively charged glutamic acid (GAG), and 2) converting the neutral residue 360, glutamine (CAG), to a positively charged histidine (CAT). We have also characterized four additional heterozygotes for the apoA-IV-3 allele. One individual was found to have the previously described substitution of lysine for glutamic acid at amino acid 230. The three other subjects had the identical mutation but at a different position in the polypeptide chain, residue 165. These results indicate that one predominant allele codes for the isoproteins apoA-IV-2 and apoA-IV-0 and that at least two major allelic variants for the isoproteins apoA-IV-1 and apoA-IV-3 are present in the population analyzed.     

Characterization of recombinant wild type and site-directed mutations of apolipoprotein C-III: lipid binding, displacement of ApoE, and inhibition of lipoprotein lipase

The physicochemical properties of recombinant wild type and three site-directed mutants of apolipoprotein C-III (apoC-III), designed by molecular modeling to alter specific amino acid residues implicated in lipid binding (L9T/T20L, F64A/W65A) or LPL inhibition (K21A), were compared. Relative lipid binding efficiencies to dimyristoylphosphatidylcholine (DMPC) were L9T/T20L > WT >K21A > F64A/W65A with an inverse correlation with size of the discoidal complexes formed. Physicochemical analysis (Trp fluorescence, circular dichroism, and GdnHCl denaturation) suggests that L9T/T20L forms tighter and more stable lipid complexes with phospholipids, while F64A/W65A associates less tightly. Lipid displacement properties were tested by gel-filtrating apoE:dipalmitoylphosphatidylcholine (DPPC) discoidal complexes mixed with the various apoC-III variants. All apoC-III proteins bound to the apoE:DPPC complexes; the amount of apoE displaced from the complex was dependent on the apoC-III lipid binding affinity. All apoC-III proteins inhibited LPL in the presence or absence of apoC-II, with F64A/W65A displaying the most inhibition, suggesting that apoC-III inhibition of LPL is independent of lipid binding and therefore of apoC-II displacement. Taken together. these data suggest that the hydrophobic residues F64 and W65 are crucial for the lipid binding properties of apoC-III and that redistribution of the N-terminal helix of apoC-III (L9T/T20L) enhances the stability of the lipid-bound protein, while LPL inhibition by apoC-III is likely to be due to protein:protein interactions.     

Apolipoprotein C-III isoforms: kinetics and relative implication in lipid metabolism

Apolipoprotein C-III (apoC-III) production rate (PR) is strongly correlated with plasma triglyceride (TG) levels. ApoC-III exists in three different isoforms, according to the sialylation degree of the protein. We investigated the kinetics and respective role of each apoC-III isoform in modulating intravascular lipid/lipoprotein metabolism. ApoC-III kinetics were measured in a sample of 18 healthy men [mean age (+/-SD) 42.1 +/- 9.5 years, body mass index 29.8 +/- 4.6 kg/m2] using a primed-constant infusion of l-(5,5,5-D3) leucine for 12 h. Mono-sialylated and di-sialylated apoC-III (apo-CIII1 and apoC-III2) exhibited similar PRs (means +/- SD, 1.22 +/- 0.49 mg/kg/day vs. 1.15 +/- 0.59 mg/kg/day, respectively) and similar fractional catabolic rates (FCRs) (0.51 +/- 0.13 pool/day vs. 0.61 +/- 0.24 pool/day, respectively). Nonsialylated apoC-III (apoC-III0) had an 80% lower PR (0.25 +/- 0.12 mg/kg/day) and a 60% lower FCR (0.21 +/- 0.07 pool/day) (P < 0.0001 for comparison with CIII1 and CIII2 isoforms). The PRs of apoC-III1 and apoC-III2 were more strongly correlated with plasma TG levels (r > 0.8, P < 0.0001) than was apoC-III0 PR (r = 0.54, P < 0.05). Finally, the PR of apoC-III2 was strongly correlated with the proportion of LDL <255 A (r = 0.72, P = 0.002). These results suggest that all apoC-III isoforms, especially the predominant CIII1 and CIII2 isoforms, contribute to hypertriglyceridemia and that apoC-III2 may play a significant role in the expression of the small, dense LDL phenotype.     

Biochemical characterization of desmosomal proteins isolated from bovine muzzle epidermis: amino acid and carbohydrate composition

The seven major desmosomal polypeptides from isolated bovine muzzle desmosomes ranging from Mr 75 000 to 250 000 were separated by gel electrophoresis, isolated and characterized with respect to their amino acid composition and sugar content. The two largest polypeptides (bands 1 and 2), i.e. desmoplakins I and II, are similar in their amino acid composition, confirming our previous immunological and biochemical data, and display a relatively high glycine content. In contrast, the other two cytoplasmic components also believed to be associated with the desmosomal plaque, i.e. polypeptides of bands 5 (Mr 83 000) and 6 (Mr 75 000), differ significantly in their amino acid composition from the desmoplakins and from each other. All four candidate polypeptides for plaque association, i.e. bands 1, 2, 5, and 6, show no significant glycosylation. The glycoproteins 4a and 4b (Mr 115 000 and 130 000) are similar in their amino acid composition, peptide analysis and immunological reactivity. Both are relatively rich in mannose and galactose but also contain sialic acid. Our determinations also indicate that the two polypeptides differ significantly in their N-acetylglucosamine and mannose content. Most, if not all, of the sugar residues are associated with a water-soluble fragment of Mr 15 500 obtained after limited digestion with V8 protease. The glycopolypeptides obtained in band 3 (Mr 164 000-175 000) are distinct from the glycopolypeptides 4a and 4b in amino acid composition, sugar content, isoelectric pH values, certain antigenic determinants and in their pattern of cleavage products obtained by treatment with proteases or cyanogen bromide. The results identify polypeptides of bands 3, 4a and 4b as glycosylated with characteristic sugar compositions. It is suggested that the major glycoproteins (bands 3, 4a, 4b) of the desmosome are integral membrane components arranged in a special way conferring resistance to detergent treatment. The possible roles of these glycoproteins in cell recognition and in adhesive functions of the desmosome are discussed.     

Human lysosomal sphingomyelinase: substrate efficacy of apolipoprotein/sphingomyelin complexes

Human apolipoprotein stimulation of sphingomyelin (SM) hydrolysis by sphingomyelinase from human skin fibroblasts has been studied. Apolipoproteins A-I, A-II, B, C-I, and E do not enhance sphingomyelin hydrolysis above control levels. In contrast, apoC-II stimulates sphingomyelin hydrolysis by approximately 2.5-fold. ApoC-III, the most potent apoprotein activator, stimulates hydrolysis by 3-4-fold. ApoC-III stimulation is not significantly different for the three different isoforms which carry 0, 1, or 2 sialic acid residues. The amino-terminal half of this apoprotein, C-III(1-40), which does not bind to phospholipid surfaces, does not activate sphingomyelinase. In contrast, the carboxyl-terminal half, C-III(41-79), which strongly binds to phospholipid surfaces, stimulates sphingomyelin hydrolysis to the same level as that produced by the intact, full-length apoprotein. Incubation of sphingomyelin vesicles with increasing proportions of apoC-III results in the formation of complexes of increasing apoC-III:SM ratio and decreasing radius. The hydrolysis of sphingomyelin in the 1:50 (mol/mol) complex was more than 2-fold greater than that of the 1:200 (mol/mol) complex. The rate of hydrolysis of egg yolk sphingomyelin in the 1:50 complex was maximal [0.9 mumol h-1 (mg of protein)-1] at the gel----liquid-crystalline phase transition temperature (Tm) of the complex (40 degrees C). The rate of hydrolysis fell markedly at either higher or lower temperature. Determination of the apparent Km and Vmax values below, at, and above Tm indicated that the temperature dependence of sphingomyelin hydrolysis was attributable primarily to changes in Vmax.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apolipoproteins C-I and C-III Inhibit Lipoprotein Lipase Activity by Displacement of the Enzyme from Lipid Droplets

Apolipoproteins (apo) C-I and C-III are known to inhibit lipoprotein lipase (LPL) activity, but the molecular mechanisms for this remain obscure. We present evidence that either apoC-I or apoC-III, when bound to triglyceride-rich lipoproteins, prevent binding of LPL to the lipid/water interface. This results in decreased lipolytic activity of the enzyme. Site-directed mutagenesis revealed that hydrophobic amino acid residues centrally located in the apoC-III molecule are critical for attachment to lipid emulsion particles and consequently inhibition of LPL activity. Triglyceride-rich lipoproteins stabilize LPL and protect the enzyme from inactivating factors such as angiopoietin-like protein 4 (angptl4). The addition of either apoC-I or apoC-III to triglyceride-rich particles severely diminished their protective effect on LPL and rendered the enzyme more susceptible to inactivation by angptl4. These observations were seen using chylomicrons as well as the synthetic lipid emulsion Intralipid. In the presence of the LPL activator protein apoC-II, more of apoC-I or apoC-III was needed for displacement of LPL from the lipid/water interface. In conclusion, we show that apoC-I and apoC-III inhibit lipolysis by displacing LPL from lipid emulsion particles. We also propose a role for these apolipoproteins in the irreversible inactivation of LPL by factors such as angptl4.     

Conformation-Determining role for the N-acetyl group in the O-glycosidic linkage, α-GalNAc-Thr

An ¹H-nmr study of 2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-D-galactopyranose (AcGalNAc) glycosylated Thr-containing tripeptides in Me₂SO-d₆ solution reveals two mutually exclusive intramolecular hydrogen bonds. In Z-Thr(AcGalNAc)-Ala-Ala-OMe, there is an intramolecular hydrogen bond between the Thr amide proton and the sugar N-acetyl carbonyl oxygen. The strength of this hydrogen bond will be dependent on the amino acid residues on the Thr C terminal side to some undetermined distance. In Ac-Thr(AcGalNAc)-Ala-Ala-OMe, a different intramolecular hydrogen bond between the sugar N-acetyl amide proton and the Thr carbonyl oxygen exists. The choice of hydrogen bonds seems dependent on the bulkiness of the residues on the Thr N terminal side. The consequence of such strong hydrogen bonds is a clearly defined orientation of the sugar moiety with respect to the peptide backbone. In the former, the plane of the sugar pyranose ring is roughly oriented perpendicularly to the peptide backbone. The latter orientation is where the plane of the sugar ring is roughly in line with the peptide backbone. In both orientations, the sugar moiety can increase the shielding of the neighboring amino acid residues from the solvent. The idea that the amino acid residues near the glycosylated Thr influence orientation of the sugar moiety with respect to the peptide backbone and in turn possibly hinder peptide backbone flexibility has interesting implications in the conformational as well as the biological role of O-glycoproteins.     

The monosaccharide composition and sequence of the carbohydrate moiety of human serum low density lipoproteins.

Human serum low density lipoprotein (d = 1.027-1.045) was delipidated with organic solvents and the apoprotein digested with thermolysin. The digest was fractionated by gel filtration and DEAE-cellulose chromatography. Two glycopeptides were obtained. One of the glycopeptides (GP-I) contained 2 residues of N-acetylglucosamine and 6 residues of mannose per mole of the glycopeptide, while the other contained 2 sialic acid, 5 mannose, 2 galactose, and 3 N-acetylglucosamine residues per mole of glycopeptide. The results of sequential enzymatic digestion with purified glycosidases, periodate oxidation, and partial acid hydrolysis lead us to propose the following sturctures for the two glycopeptides: (see article). These glycopeptides represent at least 50% of the carbohydrate moiety of LDL.     

Identification and characterization of a thermolabile antigen (TLAb, glyceraldehyde-3-phosphate dehydrogenase) in Saccharomyces cerevisiae

Five thermolabile antigens (TLAa, TLAb, TLAc, TLAd, and TLAe) have been purified from Saccharomyces cerevisiae. Recently, we reported that TLAa was identical with yeast enolase (EC 4.2.1.11). In this paper, TLAb was identified as yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) on the following bases: (1) Mr, N-terminal amino acid sequence, and isomer number of TLAb were the same as those of GAPDH; (2) anti-TLAb serum was reactive to GAPDH in the Ouchterlony test and in sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting; and (3) TLAb possessed GAPDH enzyme activity which was inhibited by anti-TLAb serum. The effect of various growth conditions on the proportion of three TLAb isoproteins (TLAb-1, TLAb-2, and TLAb-3) was examined. The proportion of two TLAb isoproteins (TLAb-1 and TLAb-2) changed depending on the cell growth phase the carbon sources, and sodium chloride shock. It is concluded that environmental stress has a differential effect on the biosynthesis of TLAb isoproteins.     

(Cu,Zn)-metallothioneins from fetal bovine liver. Chemical and spectroscopic properties

Two metallothioneins (MTs) from bovine fetal liver were purified by a combination of gel filtration and ion-exchange chromatography. The primary structures of the isoproteins MT-1 and MT-2 were elucidated by peptide and amino acid sequence analysis. The amino-terminal part was deduced from automated Edman degradations of the pyridylethylated CNBr-cleaved derivatives. The remaining part of the sequence was established by a comparison of the carboxamidomethylated tryptic peptides to those from equine liver MT-1A and MT-2B. Peptides differing in either amino acid composition or retention time from high pressure liquid chromatography were further subjected to manual Edman degradations or carboxypeptidase Y digestion. The two isoproteins consist of 61 amino acids and show a sequence identity of 90%. When compared with the primary structures of other mammalian MTs, the 20 cysteinyl residues are totally conserved, in agreement with their function as metal ligands. The two isoproteins contain Cu and Zn at a ratio of 3:4. Spectroscopic data reveal absorption properties typical for both Cu- and Zn-thiolate transitions. The marked differences of MT-1 and MT-2 in the Cu-thiolate CD features can be attributed to the six amino acid substitutions occurring exclusively in the amino-terminal parts of the molecules. It is proposed that in bovine fetal MTs also the three copper ions are preferentially bound to the first 9 cysteinyl residues (cluster B) and the four zinc ions to the remaining 11 cysteinyl residues (cluster A) suggested previously by 113Cd NMR spectroscopy of calf liver MTs (Briggs, R. W., and Armitage, I. M. (1982) J. Biol. Chem. 257, 1259-1262).     

Human plasma apolipoproteins A-IV-0 and A-IV-3. Molecular basis for two rare variants of apolipoprotein A-IV-1

Human apolipoprotein (apo) A-IV is a polymorphic plasma protein controlled by two codominant alleles at a single genetic locus. Thus far, five different isoproteins (apoA-IV-0 to apoA-IV-4) have been described in Caucasians. We have recently identified the nucleotide and amino acid substitutions that are the basis for the most common isoproteins, apoA-IV-1 and apoA-IV-2. In this report, the mutations producing the two rare isoproteins apoA-IV-0 and apoA-IV-3 are described. Analysis of the apoA-IV-0 allele revealed an insertion of 12 nucleotides in a carboxyl-terminal region, which is highly conserved among human, rat, and mouse A-IV apolipoproteins. This in-frame insertion of the 4 amino acids Glu-Gln-Gln-Gln between residues 361 and 362 of the mature protein produces the 1 charge unit more acidic apoA-IV-0 isoprotein (pI 4.92). In the apoA-IV-3 allele we identified a single G to A substitution that converts the glutamic acid (GAG) at position 230 of the mature protein to a lysine (AAG), thus adding 2 positive charge units to the apoA-IV-1 isoprotein (pI 4.97) and forming the more basic apoA-IV-3 isoprotein (pI 5.08). Comparison with the mouse and rat A-IV apolipoproteins revealed that this residue, located at position 4 of the 10th/11th amphiphilic alpha-helical repeat, is also highly conserved in evolution.     

Chemical and physical properties of human bronchial mucus glycoproteins.

Human bronchial mucus glycoproteins of different chemical types were isolated by Ecteola and gel exclusion chromatography. Chemical analysis indicated polydispersity with regard to content of sulfate and sialic acid. No blood group A, B or H activity was found in these glycoproteins. Compositions are reported for amino acid and sugar residues for several fractions obtained from both cystic fibrotic and chronic bronchitic mucus. It is noteworthy that glycoproteins extracted from a single subject contain molecules with different acid groups as well as significant differences in carbohydrate chain length.     

Sialic acids, but not their linkage to galactose residues, are required for full expression of the biological activity of human chorionic gonadotropin

One of the characteristic features of the asparagine-linked sugar chains of human chorionic gonadotropin (hCG) is that their sialic acid residues occur exclusively as the Neu5Ac alpha 2----3Gal group. In order to determine whether this sialic acid linkage is important for the functional role played by the sugar moiety of hCG or not, isomeric hCG containing the Neu5Ac alpha 2----6Gal group was prepared and its hormonal activity in vitro was investigated. On addition of the isomeric hCG to the culture medium of a murine Leydig tumor cell line, it was found that it shows the same hormonal activity as natural hCG.     

Constant and variable domains of different disaccharide structure in corneal keratan sulphate chains.

Four peptidokeratan sulphate fractions of different Mr and degree of sulphation were cut from the pig corneal keratan sulphate distribution spectrum. After exhaustive digestion with keratanase, the fragments were separated on DEAE-Sephacel and Bio-Gel P-10 and analysed for their Mr, degree of sulphation and amino sugar and neutral sugar content. It was found that every glycosaminoglycan chain is constructed of a constant domain of non-sulphated and monosulphated disaccharide units and a variable domain of disulphated disaccharide units. Total neuraminic acid of the four peptidokeratan sulphates was recovered from their isolated linkage-region oligosaccharides. In kinetic studies, the four peptidokeratan sulphates were investigated for Mr distribution after various incubation times with keratanase. There was a continuous shift towards lower Mr and no appearance of a distinct intermediate-sized product at any degradation time. The linkage-region oligosaccharide was already being liberated after a very short incubation period. From the results of these kinetic investigations in connection with the results of neuraminic acid analyses it is suggested that there exists only one disaccharide chain per peptidokeratan sulphate molecule. A model of corneal keratan sulphate is postulated. One of the alpha-mannose residues in the linkage region is bound to an oligosaccharide consisting of a lactosamine and a terminal sialic acid. The other alpha-mannose residue is attached to the disaccharide chain. This chain contains one or two non-sulphated disaccharide units at the reducing end, followed by 10-12 monosulphated disaccharide units. The disulphated disaccharide moiety of variable length is positioned at the non-reducing end of the chain.     

Purification, properties, and partial structure elucidation of a high-molecular-weight glycoprotein from cervical mucus of the bonnet monkey (Macaca radiata).

A high-molecular-weight glycoprotein has been purified from the cervical mucus of the bonnet monkey (Macaca radiata). The glycoprotein was shown to be homogeneous by electrophoresis, sedimentation equilibrium, and N-terminal group determination, and to contain 19% protein, 19% D-galactose, 18% N-acetyl-D-galactosamine, 15% N-acetyl-D-glucosamine, 11% L-fucose, 10% sialic acid, and 1% sulfate groups, corresponding to about 1800 amino acid residues and 400 carbohydrate side chains of about 9 monosaccharides. The carbohydrate chains are linked to the peptide backbone through N-acetyl-D-galactosamine and serine (or threonine) residues. Reduction with dithiothreitol and alkylation with iodoacetic acid reduced the molecular mass from 1 to 0.5 X 10(6) daltons and produced subunits having the same size, charge, and N-terminal amino acid. Electrophoretic studies suggested the presence of disulfide bonds between two chains of the glycoprotein. Degradation with alkaline borohydride gave, after fractionation on Bio-Gel P-2, fractions containing L-fucose, D-galactose, N-acetyl-D-galactosaminitol, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, and sialic acid in the ratio of 1.0:3.0:1.0:1.0:1.3:1.0. Further fractionation by electrophoresis and paper chromatography gave a charged fraction representing 13% of the original glycoprotein. Enzymic degradation and methylation studies indicated the presence of the structure alpha-Gal-(1 leads to 3)-[Fuc(1 leads to 2)]-Gal-(1 leads to 4)-GlcNAc, linked to a core component containing N-acetyl-D-galactosaminitol.