Calf Fetuin Research Articles

A Lectin Purified from Blood Red Bracket Mushroom, Pycnoporus sanguineus (Agaricomycetidae), Mycelium Displayed Affinity Toward Bovine Transferrin.

Fungal lectins constitute excellent ligands for development of affinity adsorbents useful in affinity chromatography. In this work, a lectin was purified from Pycnoporus sanguineus (PSL) mycelium using 3 procedures: by affinity chromatography, using magnetic galactosyl-nanoparticles or galactose coupled to Sepharose, and by ionic exchange chromatography (IEC). The highest lectin yield was achieved by IEC (55%); SDS-PAGE of PSL showed 2 bands with molecular mass of 68.7 and 55.2 kDa and IEC displayed 2 bands at pi 5.5 and 5.2. The lectin agglutinates rat erythrocytes, exhibiting broad specificity toward several monosaccharides, including galactose. The agglutination was also inhibited by the glycoproteins fetal calf fetuin, bovine lactoferrin, bovine transferrin, and horseradish peroxidase. The lectin was then used to synthesize an affinity adsorbent (PSL-Sepharose) and the interaction with glycoproteins was evaluated by analyzing their chromatographic behaviors. The strongest interaction with the PSL-derivative was observed with transferrin, although lower interactions were also displayed toward fetuin and lactoferrin. These results indicate that the purified PSL constitutes an interesting ligand for the design of affinity adsorbents to be used (i.e., in glycoprotein purification).

Transglycosylation of Asparagine-linked Complex-type Oligosaccharides from Glycoproteins by Endo-.BETA.-N-acetylglucosaminidase HS

Endo-β-N-acetylglucosaminidases hydrolyze N,N´-diacetylchitobiose linkages of asparagine-linked oligosaccharides. They can also cleave the linkage with suitable agents having hydroxyl groups and transfer the released oligosaccharides to the agents. Thus endo-β-N-acetylglucosaminidases are very useful for synthesis of neoglycoconjugates having asparagine-linked oligosaccharides. On the other hand, the structures of asparagine-linked oligosaccharides are divided into three groups, high-mannose type, hybrid type and complex type. We discovered a novel endo-β-N-acetylglucosaminidase (Endo), named Endo HS. Endo HS can specifically hydrolyze bi-, tri- and tetraantennary complex-type oligosaccharides from glycoproteins. We have investigated the transglycosylation reaction by Endo HS. Endo HS transferred the biantennary complex type oligosaccharide from human transferrin to p-nitrophenyl (PNP)-β-D-Glc and PNP-β-D-Gal. Endo HS was strictly distinct from other enzymes in transferring oligosaccharide to the Gal moiety. The amount of the transglycosylation product increased depending on the concentration of the acceptors. Endo HS also transferred the oligosaccharide to PNP-α-D-Glc, PNP-α-D-Gal, PNP-β-D-Man, PNP-β-D-Xyl, PNP-β-D-GlcNAc and PNP-glycerol. The amount of the transglycosylation product successively increased and became constant and then barely decreased. No apparent difference in the Km value for human transferrin as an oligosaccharide donor was observed using different acceptors such as PNP-β-D-Glc and PNP-glycerol. Endo HS also transferred the triantennary complex-type oligosaccharide from calf fetuin and the bi-, tri- and tetraantennary complex-type oligosaccharides from human α1-acid glycoprotein to PNP-β-D-Glc. In addition to glycoproteins, Endo HS transferred biantennary complex-type oligosaccharide from glycopeptides. Furthermore, Endo HS transferred bi- and triantennary complex-type oligosaccharides from glycoasparagines to various monosaccharides, oligosaccharides, sugar alcohols and glycosides. The addition of polar organic solvents was also effective for the transglycosylation efficiency. The results demonstrate that Endo HS is a useful tool for synthesis of neoglycoconjugates having a wide variety of complex-type asparagine-linked oligosaccharides from glycoproteins.

Evidence for the transglycosylation of complex type oligosaccharides of glycoproteins by endo-β- N-acetylglucosaminidase HS

Transglycosylation activity of endo-β- N-acetylglucosaminidase HS (Endo HS) was investigated using native human transferrin as a donor of an asparagine-linked oligosaccharide and p-nitrophenyl-β- d-glucose (PNP-β- d-Glc) as an acceptor of the oligosaccharide. The amount of the product increased dependent on the concentration of the acceptors. Absorption spectrum, exoglycosidase digestion and matrix assisted laser desorption and ionization-time of flight (MALDI-TOF) mass analysis of the transglycosylation product indicated that the asialobiantennary complex type oligosaccharide of human transferrin was transferred to PNP-β- d-Glc. Endo HS also transferred the oligosaccharide of human transferrin to PNP-α- d-Glc, PNP-α- d-Gal, PNP-β- d-Gal, PNP-β- d-Man, PNP-β- d-Xyl, PNP-β- d-GlcNAc, and PNP-glycerol at a different rate. No apparent difference in the K m value for human transferrin as an oligosaccharide donor was observed using different acceptors, PNP-β- d-Glc and PNP-glycerol. The amount of the transglycosylation product successively increased and became constant and then very slightly decreased during the course of enzyme reaction. Endo HS was also transferred the triantennary complex type oligosaccharide of calf fetuin and the bi-, tri-, and tetrantennary complex type oligosaccharides of human α 1-acid glycoprotein to PNP-β- d-Glc. Furthermore, Endo HS transferred an asparagine-linked oligosaccharide from a hen egg glycopeptide to PNP-β- d-Glc. The results demonstrate that Endo HS can transfer a wide variety of asparagine-linked complex type oligosaccharides to various monosaccharides. Endo HS was distinct from other enzymes in the specificity for oligosaccharide donors and acceptors.

Identification and Molecular Cloning of a Novel Glycoside Hydrolase Family of Core 1 Type O-Glycan-specific Endo-α-N-acetylgalactosaminidase from Bifidobacterium longum

We found endo-alpha-N-acetylgalactosaminidase in most bifidobacterial strains, which are predominant bacteria in the human colon. This enzyme catalyzes the liberation of galactosyl beta1,3-N-acetyl-D-galactosamine (Galbeta1,3GalNAc) alpha-linked to serine or threonine residues from mucin-type glycoproteins. The gene (engBF) encoding the enzyme has been cloned from Bifidobacterium longum JCM 1217. The protein consisted of 1,966 amino acid residues, and the central domain (590-1381 amino acid residues) exhibited 31-53% identity to hypothetical proteins of several bacteria including Clostridium perfringens and Streptococcus pneumoniae. The recombinant protein expressed in Escherichia coli liberated Galbeta1,3GalNAc disaccharide from Galbeta1,3GalNAcalpha1pNP and asialofetuin, but did not release GalNAc, Galbeta1,3(GlcNAcbeta1,6)GalNAc, GlcNAcbeta1,3GalNAc, and Galbeta1,3GlcNAc from each p-nitrophenyl (pNP) substrate, and also did not release sialo-oligosaccharides from fetuin, indicating its strict substrate specificity for the Core 1-type structure. The stereochemical course of hydrolysis was determined by (1)H NMR and was found to be retention. Site-directed mutagenesis of a total of 22 conserved Asp and Glu residues suggested that Asp-682 and Asp-789 are critical residues for the catalytic activity of the enzyme. The enzyme also exhibited transglycosylation activity toward various mono- and disaccharides and 1-alkanols, demonstrating its potential to synthesize neoglycoconjugates. This is the first report for the isolation of a gene encoding endo-alpha-N-acetylgalactosaminidase from any organisms and for the establishment of a new glycoside hydrolase family (GH family 101).

A Novel Human β1,3-N-Acetylgalactosaminyltransferase That Synthesizes a Unique Carbohydrate Structure, GalNAcβ1-3GlcNAc

We found, using a BLAST search, a novel human gene (GenBank trade mark accession number BC029564) that possesses beta3-glycosyltransferase motifs. The full-length open reading frame consists of 500 amino acids and encodes a typical type II membrane protein. This enzyme has a domain containing beta1,3-glycosyltransferase motifs, which are widely conserved in the beta1,3-galactosyltransferase and beta1,3-N-acetylglucosaminyltransferase families. The putative catalytic domain was expressed in human embryonic kidney 293T cells as a soluble protein. Its N-acetylgalactosaminyltransferase activity was observed when N-acetylglucosamine (GlcNAc) beta1-O-benzyl was used as an acceptor substrate. The enzyme product was determined to have a beta1,3-linkage by NMR spectroscopic analysis, and was therefore named beta1,3-N-acetylgalactosaminyltransferase-II (beta3GalNAc-T2). The acceptor substrate specificity of beta3GalNAc-T2 was examined using various oligosaccharide substrates. Galbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-O-para-nitrophenyl (core 2-pNP) was the best acceptor substrate for beta3GalNAc-T2, followed by GlcNAcbeta1-4GlcNAcbeta1-O-benzyl, and GlcNAcbeta1-6GalNAcalpha1-O-para-nitrophenyl (core 6-pNP), among the tested oligosaccharide substrates. Quantitative real time PCR analysis revealed that the beta3Gal-NAc-T2 transcripts was restricted in its distribution mainly to the testis, adipose tissue, skeletal muscle, and ovary. Its putative orthologous gene, mbeta3GalNAc-T2, was also found in a data base of mouse expressed sequence tags. In situ hybridization analysis with mouse testis showed that the transcripts are expressed in germ line cells. beta3GalNAc-T2 efficiently transferred GalNAc to N-glycans of fetal calf fetuin, which was treated with neuraminidase and beta-galactosidase. However, it showed no activity toward any glycolipid examined. Although the GalNAcbeta1-3GlcNAcbeta1-R structure has not been reported in humans or other mammals, we have discovered a novel human glycosyltransferase producing this structure on N- and O-glycans.

Molecular Cloning and Characterization of a Novel Human β1,4-N-Acetylgalactosaminyltransferase, β4GalNAc-T3, Responsible for the Synthesis of N,N′-Diacetyllactosediamine, GalNAcβ1–4GlcNAc

We found a novel human glycosyltransferase gene carrying a hypothetical beta1,4-glycosyltransferase motif during a BLAST search, and we cloned its full-length open reading frame by using the 5'-rapid amplification of cDNA ends method. It encodes a type II transmembrane protein of 999 amino acids with homology to chondroitin sulfate synthase in its C-terminal region (GenBank accession number AB089940). Its putative orthologous gene was also found in mouse (accession number AB114826). The truncated form of the human enzyme was expressed in HEK293T cells as a soluble protein. The recombinant enzyme transferred GalNAc to GlcNAc beta-benzyl. The product was deduced to be GalNAc beta 1-4GlcNAc beta-benzyl based on mass spectrometry and NMR spectroscopy. We renamed the enzyme beta1,4-N-acetylgalactosaminyltransferase-III (beta 4GalNAc-T3). beta 4GalNAc-T3 effectively synthesized N,N'-diacetylgalactosediamine, GalNAc beta 1-4GlcNAc, at non-reducing termini of various acceptors derived not only from N-glycans but also from O-glycans. Quantitative real time PCR analysis showed that its transcript was highly expressed in stomach, colon, and testis. As some glycohormones contain N,N'-diacetylgalactosediamine structures in their N-glycans, we examined the ability of beta 4GalNAc-T3 to synthesize N,N'-diacetylgalactosediamine structures in N-glycans on a model protein. When fetal calf fetuin treated with neuraminidase and beta1,4-galactosidase was utilized as an acceptor protein, beta 4GalNAc-T3 transferred GalNAc to it. Furthermore, the majority of the signal from GalNAc disappeared on treatment with glycopeptidase F. These results suggest that beta 4GalNAc-T3 could transfer GalNAc residues, producing N,N'-diacetylgalactosediamine structures at least in N-glycans and probably in both N- and O-glycans.

Three-Dimensional Elution Mapping of Pyridylaminated N-Linked Neutral and Sialyl Oligosaccharides

We propose a three-dimensional (3-D) sugar-mapping technique for pyridylaminated (PA) neutral and sialyl oligosaccharides as a powerful structural characterization of N-linked oligosaccharides using only picomoles of samples. The new map consists of the elution data from 42 different sialyl oligosaccharides, 26 of which are mono-, 7 of which are di-, 7 of which are tri-, and 2 of which are tetra-sialylated oligosaccharides. The 20 standard sialyl oligosaccharides were released from human serum and calf fetuin by digestion with glycoamidase A. The other 22 standard sialyl oligosaccharides were obtained by subsequent digestion of the above 20 sialyl oligosaccharides with β-galactosidase, β-N-acetylhexosaminidase, α-fucosidase, and α 2→3 specific sialidase. The present 3-D mapping method involves the following four steps: First, a neutral and sialyl PA-oligosaccharide mixture is separated by HPLC on the diethylaminoethyl (DEAE) column according to the sialic acid content, and the elution data are considered as one of the three dimensions (Z-axis). Then, neutral, mono-, di-, tri-, and tetra-sialyl oligosaccharides are individually separated on the octadecylsilyl (ODS)-silica (X-axis) and amide-silica (Y-axis) columns. The fourth step is to plot the coordinates on a two-dimensional (2-D) map. Thus, for each of the groups separated on the DEAE column, a 2-D map can be achieved. By repeating the whole process for each group of different sialylation, the layers of the 2-D map lined up on the Z-axis form a 3-D map. It is important to point out that for each neutral, mono-, di-, tri-, and tetra-sialyl oligosaccharide, the HPLC elution conditions used to obtain the X- and Y-coordinates are completely the same as described previously for neutral oligosaccharides. Furthermore, this 3-D map can clearly differentiate closely related Neu5-Acα2→6 and α2→3 containing oligosaccharides. The validity of the 3-D mapping technique was tested with sialyl PA-oligosaccharides from human IgG glycoproteins.

Inhibitory effect of calf fetuin on the cytotoxic activity of LAK cell-derived factors and tumor necrosis factor.

A protein-inhibiting LAK cell-mediated cytotoxicity was isolated from a LAK cell-conditioned medium. The N-terminal amino acid sequence of this protein appeared to be identical to fetal calf fetuin. Pure isolated fetuin, as well as commercially available preparations of this protein, were shown to inhibit cytotoxic activity of both cytotoxic proteins released by LAK cells and TNF.

Novel specificities of Mucor hiemalis endo-beta-N-acetylglucosaminidase acting complex asparagine-linked oligosaccharides.

Mucor hiemalis endo-beta-N-acetylglucosaminidase (Endo-M) was proved to act on complex type biantennary oligosaccharides of glycoproteins by using dansylated asparagine-linked and pyridylaminated oligosaccharides, as the substrate. The enzyme could act on both asialo- and sialo-biantennary oligosaccharides. This is the only endo-beta-N-acetylglucosaminidase known to act on sialo glycans, though their activity for them was weak. The enzyme could liberate complex type biantennary oligosaccharides from native human asialotransferrin, which was ascertained by a combination of the pyridylaminated method and HPLC. The enzyme had substrate specificity for high-mannose type oligosaccharides different from those of the endo-beta-N-acetylglucosaminidases of other microorganisms: ovalbumin glycopeptide-IV was a better substrate for Endo-M than glycopeptide-V. The enzyme could act on complex type triantennary oligosaccharides of dansylated glycopeptide prepared from calf fetuin. The enzyme had various novel specificities in regard to activities on complex type and high-mannose type oligosaccharides in glycoproteins.

Human salivary endo-beta-N-acetylglucosaminidase HS specific for complex type sugar chains of glycoproteins

The enzyme that catalyzed the conversion of human salivary alpha-amylase family A (HSA-A) to family B (HSA-B) was identified. It was partially purified from the precipitate obtained by centrifugation of human saliva at 105,000 x g for 60 min by solubilization with 3[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate and column chromatographies with Sephacryl S-300-HR and hydroxylapatite. The enzyme preparation was practically free from contaminating exoglycosidases and proteases. The enzyme cleaved the N,N'-diacetylchitobiose moiety of the sugar chain of HSA-A, as shown by the isolation of the protein moiety which contained 1 GlcNAc and 1 Fuc residue and the sugar chain (Gal)2(Fuc)1(GlcNAc)2(Man)3(GlcNAc). This enzyme also cleaved the N,N'-diacetylchitobiose moiety of the sugar chain of human transferrin tetraglycopeptide Asn-Tyr-Asn(GlcNAc)2(Man)3(GlcNAc)2(Gal)2-Lys to yield equimolar amounts of peptide Asn-Tyr-Asn(GlcNAc)Lys and sugar chain (Gal)2(GlcNAc)2(Man)3(GlcNAc). The enzyme was identified as an endo-beta-N-acetylglucosaminidase. The enzyme acted on HSA-A with desialylated and defucosylated outer chain moieties of the sugar chains at a similar rate as that of native HSA-A. The enzyme activity was reduced to 13 and 5% using HSA-A with the sugar chains whose outer chain moieties lacked Gal and GlcNAc, respectively, from the nonreducing end. The enzyme also acted on human transferrin, calf fetuin, and asparagine oligosaccharides of transferrin and fetuin. On the other hand, the enzyme did not act on ovalbumin, RNase B, Taka-amylase, yeast invertase, and ovalbumin asparagine oligosaccharides. These results indicate that human salivary endo-beta-N-acetylglucosaminidase is specific for complex type sugar chains and can release the sugar chains from native glycoproteins and glycopeptides regardless of the existence of a Fuc residue on the proximal GlcNAc of the N,N'-diacetylchitobiose core of their sugar chains. The source of the enzyme was epithelial cells peeling from the oral cavity epithelium into saliva. The enzyme was thought to be integrated on the surface of the epithelial cell membrane. This enzyme was named endo-beta-N-acetylglucosaminidase HS. Thus, these studies indicate that the properties of the enzyme are distinct from those of known endo-beta-N-acetylglucosaminidase and endo-beta-N-acetylglucosaminidase HS is a novel endo-beta-N-acetylglucosaminidase.

Adherence of Helicobacter pylori cells and their surface components to HeLa cell membranes

Four Helicobacter pylori strains were used to develop in vitro methods to assess adherence to HeLa cells. Using direct detection by microscopy, adhesion scores increased with the initial bacteria-to-cell ratio. The urease method assessed H. pylori bound to HeLa cells by their urease activity. The percentage of the original inoculum adhering to HeLa cells remained constant for initial ratios from 10 2 to 10 5 bacteria per cell. An ELISA using anti- H. pylori serum assessed whole bacteria or components bound to HeLa cell fractions. By all three methods, the four H. pylori strains were adherent to HeLa cells or membranes whereas Campylobacter fetus and Providencia control strains were not. The adherence of H. pylori whole cells decreased following extraction with saline, water, or glycine buffer and most of the superficial adhering material (SAM) was present in the saline or water extracts. SAM bound better to HeLa membranes than to calf fetuin or bovine serum albumin (BSA); binding was inhibited by preincubation of SAM with HeLa membranes but not with fetuin or BSA or by pretreatment of HeLa membranes with neuraminidase. These data indicate that SAM has a specific receptor on the HeLa cell membranes. By gel exclusion chromatography of bacterial extracts, the most adherent components were found in the fractions which also contained the highest urease activity; these fractions included urease subunit antigens. We conclude that adherence of H. pylori can be assessed by microtiter assays and involves bacterial surface material which co-purifies with urease and is different from the N-acetyl-neuraminyl-lactose binding hemagglutinin.

Production, purification, and characterization of Neurospora sitophila lectin.

Neurospora sitophila produced extracellular and cell wall-associated lectins. The addition of l-sorbose to a culture resulted in a decrease in the production of the former lectin and complete abolition of the latter. The lectin in the culture filtrate was purified by bovine submaxillary mucin-conjugated Sepharose chromatography. The molecular weight of the lectin was calculated to be approx. 40,000 by Sephacryl S-200 gel filtration, and that of the subunit to be approx. 22,000 by SDS/polyacrylamide- gel electrophoresis. The lectin was not inhibited by simple sugars or their homopolymers. It was inhibited strongly by glycoproteins from human erythrocyte membrane and bovine submaxillary mucin, and moderately by α1-acid glycoprotein from human plasma, human IgA and IgM, and fetal calf fetuin. The lectin agglutinated human type A, B and O erythrocytes to the same degree. Erythrocytes from chick, horse, rabbit and sheep were more efficiently agglutinated.

A two-dimensional 1H-n.m.r. (500 MHz) and 13C-n.m.r. (125 MHz) study of N-linked glycopeptides derived from calf fetuin

The oligosaccharide part of an N-linked triantennary glycopeptide from calf fetuin with fourteen carbohydrate residues and its smaller derivatives obtained by successive enzymic cleavage of the terminal residues were investigated using 2D 1H-n.m.r. (500 MHz) and 13C-n.m.r. (125 MHz) spectroscopy. Assignments have been made of the resonances of almost all the protons of the constituent carbohydrate residues in these glycopeptides. A comparison of the 1H chemical shifts and coupling constants, as determined from the cross-peaks, has shown the dependence of these parameters on the interactions of spatially related neighbouring carbohydrates. Small conformational changes take place upon elongation of the oligosaccharide side-chains.

Structural and NMR assignment study of the O‐linked glycopeptides of calf fetuin

AbstractTwo O‐linked glycopeptides were isolated by protease digestion of calf fetuin. Using 13C and 1H NMR spectroscopy, it was established that both carry identical trisaccharide side‐chains, but they were found to possess different peptide moieties. Therefore, they must originate from different regions of the polypeptide backbone of fetuin. The two glycopeptides carry a total of three oligosaccharide chains, in accordance with previously published data. One glycopeptide was found to carry two side‐chains: one is linked to a serine and the other to a threonine that are separated by one alanine only. The other glycopeptide carries one sugar chain linked to a serine residue. It was not possible to isolate any glycopeptide which contained a second sialic acid residue directly linked to the N‐acetylgalactosamine, as reported earlier. Some long‐range chemical shift effects were observed in the NMR spectra of the glycopeptide, indicating a specific conformation for this glycopeptide as a result of the proximity of the two oligosaccharide side‐chains.

Conformational analysis and the fine structure of cross peaks in phase‐sensitive homonuclear two‐dimensional correlated NMR spectra of oligosaccharides

Useful structural and conformational information was obtained from a detailed analysis of the fine structure of cross peaks that appears in the phase-sensitive homonuclear correlated two-dimensional spectra of complex large glycopeptides. The small chemical shift range observed for the oligosaccharide part results in a variety of cross-peak patterns. A catalogue of cross-peak fine structures and patterns expected for common carbohydrates are presented which can help in making some, all-important and crucial, proton resonance assignments. Analysis of the vicinal coupling constants extracted from the cross peaks corresponding to the hydroxymethyl protons of the inner core carbohydrates of glycopeptides derived from calf fetuin, has yielded information regarding the time-averaged solution conformation of this triantennary structure. It was shown that in solution the Man(alpha 1-6) arm of the core pentasaccharide unit, which carries a single antenna, adopts for an appreciable part of the time a conformation which brings it close to the arm that is attached to the peptide part. A similar conformation for the Man(alpha 1-6) arm was previously observed for the biantennary oligosaccharide chain. Therefore, we concluded that the presence of an additional antenna on the Man(alpha 1-3) arm of the core unit in the triantennary structure does not alter the basic biantennary conformation.

One- and two-dimensional 90.5-MHz 13C-NMR spectroscopy of the N-linked triantennary oligosaccharide units of calf fetuin

Complete assignments of all anomeric resonances in the proton and carbon spectra of the N-linked oligosaccharide units of fetuin were made using one- and two-dimensional NMR spectroscopy. We are able to confirm the presence of microheterogeneity in the N-acetylneuraminic acid linkages to the galactose residues and the presence of a unique triantennary structure which carries a side chain: NeuAcα(1–3)Galβ(1–3) GlcNacβ(1–4)-. Anomeric carbon chemical shifts changes resulting from long-range conformational effects were observed.

Natural-Abundance, 13C-Nuclear Magnetic Resonance-Spectral Studies of Carbohydrates Linked to Amino Acids and Proteins

This chapter presents the 13 C-nuclear magnetic resonance-spectral studies of carbohydrates linked to amino acids and proteins. This technique is dealt with in terms of the information that may be gained about the glycoprotein without its modification, which would lead to the destruction of the native sample and possible microheterogeneity of the oligosaccharide chains. The covalent attachment of carbohydrates to amino acid residues of glycoproteins falls into three main categories including the glycosidic ( O -glycosyl) linkage, the N -glycosyl linkage, and the S -glycosyl linkage. The analysis of the anomeric-carbon region of two glycoproteins— human α 1 -acid glycoprotein (α 1 -AGP) and calf fetuin—provides additional information not available by more conventional methods. Any structural analysis of such a complex system must be based on the assignments of the various signals in the spectra. Their spectra shows that resonances corresponding to inner carbohydrate display residues a much larger line-width than resonances corresponding to outer residues. This reflects the relative mobility of such residues and aids in distinguishing between terminal residues and inner residues of the intact oligosaccharide chains of intact glycoproteins. The structural analysis of glycopeptides and glycoprotein containing O -glycosyl linkages is also elaborated in the chapter.

NH 2-terminal sequence of Calf Fetuin

Calf fetuin, one of the 3 major known fetal proteins has been isolated by a two-step purification procedure and characterized by aminoacid composition. The purified glycoprotein, which consisted of a single chain, was submitted to 47 steps of automatic aminoacid sequencing, allowing to determine 44 positions. This section of the molecule was devoid of carbohydrates. Comparison of this sequence with a variety of detectable potentially related protein did not allow to point to any detectable homology.

Glycorprotein biosynthesis in calf kidney glycoprotein sialytransferase activitities towards serum glycoproteins and calf Tamm-Horsfall glycoprotein

CMP-AcNeu:glycoprotein sialyltransltransltransltransltransferase of calf kidney cortex was characterized using serum glycoproteins and Tamm-Horsfall glycoprotein, obtained from calf urine, as acceptors. Native calf Tamm-Horsfall glycoprotein showed the best acceptor properties, followed by desialylated calf fetuin and desialylated human alpha 1-acid glycoprotein exhibiting V values of, respectively, 114, 63 and 41 nmol/h per g wet wt. of kidney cortex and Km values of 0.12, 0.16 and 0.26 mM glycoprotein acceptor. Desialylated ovine submaxillary mucine appeared to be a very poor acceptor. Tamm-Horsfall glycoprotein sialyltransferase could be distinguished from serum glycoprotein sialyltransferase by competition studies. In addition the two glycoprotein sialyltransferase activities showed different distributions over the three regions of the calf kidney: the ratios of the Tamm-Horsfall to serum glycoprotein sialyltransferase activities decreased from 3.3 in the cortex to 0.8 and 0.4 in the medulla and the papilla, respectively. It was concluded that in calf kidney at least two different sialyltransferases exist. The high cortical Tamm-Horsfall glycoprotein sialyltransferases activity corresponds markedly to the origin of the urinary Tamm-Horsfall glycoprotein, namely the distal part of the kidney tubule. Inactivation of glycoprotein sialyltransferase activity by preincubation at various temperatures and during storage at 0 degree C, could be reduced by the addition of CMP-AcNeu. The possible relevance towards the in vivo sialylation of this finding is discussed.

Sialyltransferase in fetal tissues: Incorporation of sialic acid into endogenous and exogenous glycoprotein acceptors

The properties of exogenous sialyltransferase activity in fetal mouse liver were compared with the endogenous enzyme activity. Nonionic detergents such as Tergitol inhibited endogenous activity but released the enzyme from the 30,000 g particulate fraction and allowed it to react with the exogenous glycoprotein acceptors, partially desialylated α-fetoprotein and calf fetuin. Zinc ions partially transferred the enzyme activity from the particulate fraction to the supernatant, while incubation at 45 °C preferentially inhibited endogenous enzyme activity. α-Fetoprotein, sialylated by the endogenous enzyme preparation, was determined to be 7% of the total incorporation of radioactivity by immunoprecipitation with mouse anti-α-fetoprotein and subsequent sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. Sialyltransferase activity in fetal liver, yolk sac, placenta and embryo (minus liver) was measured during development. Exogenous enzyme activity of liver in early development (day 13.5) was twice as high as that in yolk-sac tissues that synthesize α-fetoprotein. Furthermore, enzyme activity increased 12-fold in fetal liver and 4-fold in yolk sac by day 18.5 of development. Activity in placenta increased 2.9-fold and then decreased, while embryo (minus liver) showed no increase. Endogenous enzyme activity increased temporally in these tissues, but the increase was less than that measured by using exogenous acceptors.