The acid and enzymic hydrolysis of O-acetylated sialic acid residues from rabbit Tamm–Horsfall glycoprotein

Rabbit Tamm-Horsfall glycoprotein and bovine submaxillary glycoprotein were both found to contain sialic acid residues which are released at a slow rate by the standard conditions of acid hydrolysis. These residues are also resistant to neuraminidases from Vibrio cholerae and Clostridium perfringens. This behaviour was attributed to the presence of O-acetylated sialic acid, since the removal of O-acetyl groups by mild alkaline treatment normalized the subsequent release of sialic acid from rabbit Tamm-Horsfall glycoprotein by acid and by enzymic hydrolysis. Determination of the O-acetyl residues in rabbit Tamm-Horsfall glycoprotein indicated that on average two hydroxyl groups of sialic acid are O-acetylated, and these were located on the polyhydroxy side-chain of sialic acid or on C-4 and C-8. These findings confirm the assumption that certain O-acetylated forms of sialic acid are not substrates for bacterial neuraminidases. Several explanations have been suggested to explain the effect of O-acetylation of the side-chain on the rate of acidcatalysed hydrolysis of sialic acid residues.     

Guinea-pig kidney beta-N-acetylgalactosaminyltransferase towards Tamm-Horsfall glycoprotein. Requirement of sialic acid in the acceptor for transferase activity.

A beta-N-acetylgalactosaminyltransferase that preferentially transferred N-acetylgalactosamine to Sd(a-) Tamm-Horsfall glycoprotein was found in guinea-pig kidney microsomal preparations. This enzyme was kidney-specific and was able to transfer the sugar to other glycoproteins, such as fetuin and alpha 1-acidic glycoprotein. The presence of sialic acid in the acceptors was essential for the transferase activity when either glycoproteins or their Pronase glycopeptides were used as acceptors. Two glycopeptides (Tamm-Horsfall glycopeptides I and II) with a different carbohydrate composition were separated by DEAE-Sephacel chromatography from Pronase-digested Tamm-Horsfall glycoprotein. The amount of N-acetylgalactosamine transferred to glycopeptides by the enzyme correlated with their degree of sialylation. Enzymic digestion of N-[14C]acetylgalactosamine-labelled Tamm-Horsfall glycopeptide II showed that the transferred sugar was susceptible to beta-N-hexosaminidase. The amount of sugar cleaved by beta-hexosaminidase was strongly increased when the labelled Tamm-Horsfall glycopeptide II was pretreated with mild acid hydrolysis, a procedure that removed the sialic acid residues. Alkaline borohydride treatment of the labelled Tamm-Horsfall glycopeptide II did not release radioactivity, thus indicating that enzymic glycosylation took place at the N-asparagine-linked oligosaccharide units of Tamm-Horsfall glycoprotein.     

Preparation and chemical characterisation of calf Tamm-Horsfall glycoprotein.

Tamm-Horsfall glycoprotein preparations were obtained from calf urine by 1.0 M NaCl precipitation followed by 4 M urea/Sepharose 4B chromatography. By using 0.1% sodium dodecyl sulfate polyacrylamide gel electrophoresis a molecular weight of 86 500 +/- 4500 (n = 12) was calculated for the glycoprotein. Amino acid and carbohydrate analyses were performed, the carbohydrate composition being (in residues per 100 amino acid residues in the glycoprotein): fucose, 0.90; galactose, 4.82; mannose, 4.63;N-acetylglucosamine, 7.36; N-acetylgalactosamine, 1.38; sialic acid, 2.93. Under conditions of mild acid hydrolysis (0.05 M H2SO4, 80 degrees C, 1 h) the calf Tamm-Horsfall glycoprotein preparations were degraded partially into two lower molecular weight fragments (approximate Mr 66 000 and 51 000), as shown by polyacrylamide gel electrophoresis, both fragments being periodic acid-Schiff reagent positive.     

A new sialic acid analogue, 9-O-acetyl-deaminated neuraminic acid, and alpha -2,8-linked O-acetylated poly(N-glycolylneuraminyl) chains in a novel polysialoglycoprotein from salmon eggs

A new polysialoglycoprotein, designated PSGP(On), was isolated from the unfertilized eggs of the kokanee salmon, Oncorhynchus nerka adonis. 400-MHz 1H NMR analyses showed the O. nerka adonis PSGP contained alpha -2,8-linked oligo- and polysialic acid (polySia) chains that were made up of 4-O-Ac-, 7-O-Ac-, and 9-O-Ac esters of N-glycolylneuraminic acid (Neu5Gc) residues. The presence of a new sialic acid derivative, identified by 1H NMR as 9-O-acetyl-2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (trivial name, 9-O-acetyldeaminated neuraminic acid; 9-O-Ac-KDN), was also shown to be present as a minor component. The O-acetylated KDN residues appear to cap the nonreducing termini of the O-acetylated poly(Neu5Gc) chains. The O-acetylated polySia chains were resistant to depolymerization by bacterial exosialidases and a bacteriophage-derived endo-N-acylneuraminidase that is specific for catalyzing the hydrolysis of alpha -2,8-linkages in polySia containing either N-acetylneuraminic acid or Neu5Gc residues. After de-O-acetylation by mild alkali, the polySia chains were sensitive to digestion by endo-N-acylneuraminidase, yet partially resistant to exosialidase. These data confirm the alpha -2,8-ketosidic linkage in these chains and the nonreducing terminal location of the KDN residues. These results extend further the range of structural diversity in polySia-containing glycoconjugates, and in the family of naturally occurring sialic acids. They also suggest that the O-acetylated Neu5Gc and 9-O-Ac-KDN residues may have an important role during oogenesis.     

Analytical detection of 9(4)-O-acetylated sialoglycoproteins and gangliosides using influenza C virus.

The unique glycoprotein of influenza C virus, designated hemagglutinin (HEF), exhibits three functions: hemagglutination, esterase activity, and fusion factor. As the virus uses 9-O-acetylated sialic acid as a high-affinity receptor determinant for attachment to cells, its binding activity was used to reveal O-acetylated sialic acid residues after polyacrylamide gel electrophoresis and transfer onto nitrocellulose sheets of proteins and thin-layer chromatography of lipids. The specificity of the binding for O-acetylated sialoglycoconjugates was investigated. Our results showed that influenza C virus could detect the different forms of the two murine glycophorins which are known to be O-acetylated sialoglycoconjugates. The virus also bound to O-acetylated gangliosides isolated from embryonic chicken brain such as purified O-acetylated NeuAc alpha (2-8)NeuAc alpha (2-8)NeuAc alpha (2-3)Gal beta (1-4)Glc beta (1-1)ceramide (GT3). The esterase activity of the HEF protein of influenza C virus was used to unmask the sialic acid. After its deacetylation by the virus enzyme, the O-acetylated GT3 was recognized by a monoclonal antibody which binds only to the nonacetylated derivative. The results presented here show that influenza C virus is a discriminating analytical probe for identifying O-acetylated sialoglycoconjugates directly after Western blotting of proteins and thin-layer chromatography of lipids, thus providing a new analytical tool.     

The turnover rate of rabbit urinary Tamm–Horsfall glycoprotein

1. The turnover rate of urinary Tamm-Horsfall glycoprotein in rabbits was determined by two different methods. The first involved measurement of the pool size of the glycoprotein in rabbit kidney and the daily urinary excretion rate by a radioimmunoassay from which the turnover rate was calculated. 2. The second method made use of the incorporation in vivo of Na(2) (14)CO(3) and sodium [(14)C]acetate. After a single intramuscular injection of one of these compounds, urine collections were made every 24h and the glycoprotein was isolated and its specific radioactivity was determined. 3. Incorporation of the label into urinary HCO(3) (-), urea and plasma fibrinogen was also examined. The specific radio-activities of the O-acetyl, sialic acid, aspartic acid and glutamic acid residues isolated from the Tamm-Horsfall glycoprotein were compared and their half-lives were compared with that of the intact glycoprotein. The two methods gave results in quite close agreement and indicated a half-life for the glycoprotein of approx. 9h. 4. An attempt was made to localize the glycoprotein within the kidney and within the cell. It is present throughout the kidney, but was not detected in the brush-border fraction isolated from the proximal tubules. From differential cell-centrifugation studies, the glycoprotein seemed to be predominantly present in the soluble fraction (100000g supernatant). This suggests that it is either largely a soluble cytoplasmic component or is very loosely bound to a membrane, being readily released under the gentlest homogenization procedure. 5. The half-life of Tamm-Horsfall glycoprotein in human kidney was found by the radioimmunoassay method to be approx. 16h. The similarity between the composition of Tamm-Horsfall glycoprotein and human erythropoietin is discussed.     

Morphological and conformational studies of Tamm-Horsfall urinary glycoprotein

We have studied the circular dichroic properties of normal and cystic fibrotic Tamm-Horsfall urinary glycoproteins, and the asialo-derivatives (ca. 80% removal of sialic acid with neuraminidase). There was no evidence of α-helicity in Tamm-Horsfall urinary glycoprotein, but the results do indicate a significant amount of β-structure. The circular dichroic spectra of normal and cystic fibrotic Tamm-Horsfall urinary glycoproteins and the asialo-derivatives were identical, thus suggesting that there is no major difference in the ordered secondary structure of Tamm-Horsfall urinary glycoprotein in cystic fibrosis (relative to normal Tamm-Horsfall urinary glycoprotein), and that sialic acid exerts no major effect on the β-structure. Also, the circular dichroic spectrum of Tamm-Horsfall urinary glycoprotein was not affected by Ca²⁺ at concentrations just below that required for gel formation. Electron microscopic studies reveal the presence of a supramolecular helical structure arising from subunit interactions. This structure was characterized by a repeat of 120–130 Å and a minimal helix diameter of ca. 40 Å, although this value varied depending on the number of interacting helices. The helical structure was observed for normal, cystic fibrotic, and asialo derivatives of Tamm-Horsfall urinary glycoproteins, and was independent of added Ca²⁺. Guanidine hydrochloride treatment, followed by dialysis, irreversibly destroyed this supra-molecular helical structure, but the β-structure was partially restored, as indicated by the circular dichroic spectrum. The Ca²⁺-mediated gel formation was found to be inhibited in asialo-Tamm-Horsfall urinary glycoprotein.     

Developmental Changes in the Biosynthesis of Sialoglycoprotein Substrates for Intrinsic Synaptic Sialidase

Abstract: N′-Acetyl-d -[6-³H]mannosamine was administered to 13- and 28-day-old rats by intraventricular injection. At various time intervals following the injection, synaptic membranes were prepared and the incorporation of radiolabel into sialic acid residues released from endogenous glycoproteins and gangliosides by intrinsic sialidase determined. Radiolabel was incorporated into synaptic membrane gangliosides and glycoproteins, and at all times tested, >90% of the label was associated with sialic acid. Sialic acid released from endogenous glycoproteins by intrinsic sialidase present in 28-day membranes incorporated only 20–25% as much radiolabel per nmole as sialic acid released by mild acid hydrolysis or by exogenous neuraminidase. In contrast, sialic acid released from glycoproteins present in 13-day-old membranes by intrinsic sialidase, mild acid hydrolysis, or exogenous neuraminidase all were similarly labelled. At both ages the specific radioactivity (cpm/nmol) of sialic acid released from gangliosides by the intrinsic enzyme was similar to the total ganglioside sialic acid released by mild acid hydrolysis. The results identify glycoprotein substrates for intrinsic synaptic membrane sialidase as a distinct metabolic class in the mature brain and suggest the occurrence of a developmentally related change in the metabolism of these glycoproteins.     

An azidoaryl thioglycoside of sialic acid. A potential photoaffinity probe of sialidases and sialic acid-binding proteins

An azidoaryl thioglycoside of sialic acid was prepared, as a potential photoaffinity probe reagent for the analysis of sialidases and sialic acid-binding proteins, by treatment of the glycosyl chloride of N-acetylneuraminic acid methyl ester with potassium thioacetate to give, in 70% yield, methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-2-S-acetyl-2,3,5-trideoxy-2-thio-alph a-D- glycero-D-galacto-2-nonulopyranosonate. Selective hydrolysis of the thioacetate ester, followed by condensation with 4-fluoro-3-nitrophenyl azide, O-deacetylation, and hydrolysis gave (4-azido-2-nitrophenyl)- 5-acetamido-2,3,5-trideoxy-2-thio-alpha-D-glycero-D-galacto-2- nonulopyranosidonic acid.     

Canine pulmonary angiotensin-converting enzyme. Physicochemical, catalytic and immunological properties.

Antiontensin-converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) has been solubilized from canine pulmonary particles and purified to apparent homogeneity. A value of approx. 140000 was estimated for the molecular weight of the native and the reduced, denatured forms of the enzyme. No free NH2-terminal residue was detected by the dansylation procedure. Carbohydrate accounted for 17% of the weight of the enzyme, and the major residues were galactose, mannose and N-acetylglucosamine with smaller amounts of sialic acid and fucose. Removal of sialic acid residues with neuraminidase did not alter enzymatic activity. The enzyme contained one molar equivalent of zinc. Addition of this metal reversed stimulation and inhibition of activity observed in the presence of Co2+ and Mn2+, respectively. Immunologic homology of pure dog and rabbit enzymes was demonstrable with goat antisera. Fab fragments and intact IgG antibodies displayed similar inhibition dose vs. response curves with homologous enzyme, whereas the fragments were poor inhibitors of heterologous activity compared to the holoantibodies. The canine glycoprotein was much less active than the rabbit preparation in catalyzing hydrolysis of Hip-His-Leu. In contrast, the two enzymes exhibited comparable kinetic parameters with angiotensin I as substrate.     

Rabbit urinary tamm-horsfall glycoprotein. Chemical composition and tentative carbohydrate structure

The urinary Tamm-Horsfall protein (THP) is the major glycoprotein secreted by the mammalian kidney. We recently isolated and immortalized thick ascending limb of Henle cells from rabbit kidney, which produce Tamm-Horsfall protein in cell culture in vitro. In order to further study the yet undefined functional role and biosynthetic pathways of this protein, we first re-examined the chemical composition and the carbohydrate structure of rabbit urinary Tamm-Horsfall protein. Using precipitation with 0.58 mol/l NaCl a protein was isolated from rabbit urine which showed extensive microheterogeneity and had an average molecular mass of 95 kDa. Deglycosylation of the protein led to a loss of microheterogeneity and yielded a molecular mass of 58-60 kDa. Amino-acid analysis of the native and deglycosylated protein revealed a lower cysteine (20 mol/mol THP) and a higher histidine (20 mol/mol THP) content than described previously. Chemical analysis of the carbohydrates showed a high glucosamine (50 mol/mol THP), galactose (43 mol/mol THP), and mannose (24 mol/mol THP) content. The amount of sialic acid was 15 mol/mol THP. Using lectins to identify the structure of the carbohydrate chains it was shown that rabbit Tamm-Horsfall protein possesses complex-type oligosaccharide chains with terminal sialic acid, beta-galactose, and probably alpha-fucose and chains of the mucin type. These results indicate that some of the cysteine residues in the polypeptide chain of THP can be replaced by histidine, suggesting a role of some cysteins in metal binding rather than intramolecular stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sialic acid is selectively involved in the interaction of agonists with M2 muscarinic acetylcholine receptors

Neuraminidase and slight acid hydrolysis were used to investigate the role of sialic acid residues in the binding of muscarinic agonists and antagonists to membranes from tissues rich in M1 and M2 receptors. Membranes were pretreated with neuraminidase at pH 5 and the binding parameters were determined from competitive experiments with (3H)-quinuclidinylbenzylate. The removal of sialic acid residues reduced the affinity of muscarinic agonists for cerebellum, heart and lung membranes (M2), in contrast to striatum (M1). The affinity of antagonists was not affected. Thus, sialic acid is selectively involved in the interaction of agonists with M2 muscarinic receptors.     

On the role of sialic acid in the rheological properties of mucus.

The importance of sialic acid in the rheological properties of mucus has been investigated. Both bovine cervical mucus, which is a gel, and the structural glycoprotein derived from it were studied before and after treatment with neuraminidase which selectively cleaves terminal sialic acid residues. The storage modulus, viscosity and circular dichroism spectrum were all essentially changed after removal of the sialic acid. These results would indicate that removal of sialic acid does not affect the physical structure of the glycoprotein and it is concluded that sialic acid has no significant role in the rheological properties of cervical mucus.     

The influence of sialic acid residues on the ability of glycoproteins toi nteract electrostatically with chondromucoprotein

1. Although glycoproteins with less than 1% of sialic acid (fibrinogen, lipoproteins, gamma-globulins) interact electrostatically with chondromucoprotein to form insoluble complexes, interaction with glycoproteins containing larger amounts of sialic acid (orosomucoid, urine glycoprotein, seromucoid, fraction VI) was electrostatically impossible. Reasons for this are discussed. 2. The latter glycoproteins interacted with chondromucoprotein after mild acid hydrolysis or neuraminidase treatment, complex-formation being inversely related to their sialic acid content. 3. Complex-formation with sialic acid-deficient orosomucoid was maximum at pH3.6 and negligible above its isoelectric point of pH5, and was inhibited by Ca(2+) ions and EDTA. 4. These results are discussed in relation to the carbohydrate composition and biological activities of euglobulin fractions, and of complexes formed by adding chondromucoprotein to abnormal plasmas which may contain sialic acid-deficient glycoproteins owing to faulty carbohydrate metabolism.     

Infection of ciliated cells by human parainfluenza virus type 3 in an in vitro model of human airway epithelium

We constructed a human recombinant parainfluenza virus type 3 (rPIV3) that expresses enhanced green fluorescent protein (GFP) and used this virus, rgPIV3, to characterize PIV3 infection of an established in vitro model of human pseudostratified mucociliary airway epithelium (HAE). The apical surface of HAE was highly susceptible to rgPIV3 infection, whereas only occasional cells were infected when virus was applied to the basolateral surface. Infection involved exclusively ciliated epithelial cells. There was little evidence of virus-mediated cytopathology and no spread of the virus beyond the ciliated cell types. Infection of ciliated cells by rgPIV3 was sensitive to a neuraminidase specific for alpha2-6-linked sialic acid residues, but not to a neuraminidase that cleaves alpha2-3- and alpha2-8-linked sialic acid residues. This provided evidence that rgPIV3 utilizes alpha2-6-linked sialic acid residues for initiating infection, a specificity also described for human influenza viruses. The PIV3 fusion (F) glycoprotein was trafficked exclusively to the apical surface of ciliated cells, which also was the site of release of progeny virus. F glycoprotein localized predominately to the membranes of the cilial shafts, suggesting that progeny viruses may bud from cilia per se. The polarized trafficking of F glycoprotein to the apical surface also likely restricts its interaction with neighboring cells and could account for the observed lack of cell-cell fusion. HAE derived from cystic fibrosis patients was not more susceptible to rgPIV3 infection but did exhibit limited spread of virus due to impaired movement of lumenal secretions due to compromised function of the cilia.     

Release of sialic acid alters the stability of the membrane potential in cardiac muscle

Intracellular recording techniques and neuraminidase, an enzyme that specifically catalyzes the hydrolysis of sialic acid's glycosidic linkage in glycoproteins and glycolipids, were employed to investigate the role of sialic acid residues in maintaining a stabilized resting potential or rhythmic electrical activity in embryonic chick cardiac muscle. Free sialic acid was quantified by a fluorometric assay. Release of more than 25% of the sarcolemma-bound sialic acid from spheroidal aggregates of cultured heart cells resulted in a) depolarizing fluctuations in the membrane potential, b) initiation of spontaneous firing in the presence of tetrodotoxin, c) arrhythmic spontaneous activity, d) depolarization of the maximum diastolic potential, and e) a significant reduction in the plateau and duration of the action potential. Control experiments demonstrated that these effects were not caused by phospholipase contamination of the enzyme or by the sialic acid released during hydrolysis.     

Use of radioactive glucosamine in the perfused rat liver to prepare alpha 1-acid glycoprotein (orosomucoid) with 3H- or 14C-labelled sialic acid and N-acetylglucosamine residues.

1. A method was developed whereby [1-14C]glucosamine was used in a perfused rat liver system to prepare over 2 mg of alpha 1-acid glycoprotein with highly radioactive sialic acid and glucosamine residues. 2. The liver secreted radioactive alpha 1-acid glycoprotein over a 4-6 h period, and this glycoprotein was purified from the perfusate by chromatography on DEAE-cellulose at pH 3.6. 3. The sialic acid on the isolated glycoprotein had a specific radioactivity of 3.1 Ci/mol, whereas the glucosamine-specific radioactivity was 4.3 Ci/mole. The latter amino-sugar residues on the isolated protein were only 13-fold less radioactive than the initially added [1-14C]glucosamine. Orosomucoid with a specific radioactivity of 31.3 microCi/mg of protein was obtainable by using [6-3H]glucosamine. 4. The amino acid composition of the purified orosomucoid was comparable with that found by others for the same glycoprotein isolated from rat serum. A partial characterization of the carbohydrate structure was done by sequential digestion with neuraminidase, beta-D-galactosidase and beta-D-hexosaminidase. 5. Many other radioactive glycoproteins were found to be secreted into the perfusate by the liver. Thus this experimental system should prove useful for obtaining other serum glycoprotein with highly radioactive sugar moieties.     

Sialic acid and neuraminidase activity in the frog oviduct: comparative biochemical investigation in the different tracts during the reproductive cycle

1. A biochemical study was carried out on the protein-bound and lipid-bound sialic acid, and neuraminidase activity in the different tracts of the oviduct of the frog Rana esculenta during the reproductive cycle. 2. Plasma sexual steroids were also investigated by RIA. 3. Fluctuations in neuraminidase activity are related to that of glycoprotein sialic acid and plasma estradiol. Glycolipid sialic acid does not have a close relationship either with neuraminidase or plasma estradiol. 4. Very high plasma concentration of progesterone before ovulation and, on the contrary, its drop after ovulation were observed. 5. The results are discussed and hypotheses advanced to explain fluctuations of the studied parameters during the reproductive cycle.     

Enzymatic properties of neuraminidases from Arthrobacter ureafaciens.

Neuraminidase I and neuraminidase II from Arthrobacter ureafaciens were characterized. As determined by gel filtration on Ultrogel AcA 44, the molecular weights of neuraminidases I and II were 51,000 and 39,000, respectively. Neuraminidases I and II were similar to each other in their enzymatic properties except for the substrate specificities towards gangliosides and erythrocyte stroma. Their optimal pHs were between 5.0 and 5.5 with N-acetylneuraminosyl-lactose or bovine submaxillary mucin as substrates, but with colominic acid as a substrate, the pH optimum was between 4.3 and 4.5. They were most active around 53 degrees C, were stable between pH 6.0 and 9.0, and were thermostable up to 50 degrees C. They did not require Ca2+ for activity and were not inhibited by EDTA. They were inhibited only slightly or not at all by p-chloromercuribenzoic acid of Hg2+. Both neuraminidases I and II were able to hydrolyze the alpha-ketosidic linkage of N-glycolylneuraminic acid as well as that of N-acetylneuraminic acid, and were able to liberate substantially all of the sialic acid from various kinds of substrates. However, they cleaved only about 50% of the sialic acid from bovine submaxillary mucin. The saponification of bovine submaxillary mucin by mild alkali treatment, on the other hand, resulted in an increased susceptibility to the neuraminidases and brought about the complete liberation of sialic acid. Remarkable differences were observed between neuraminidases I and II as regards substrate specificities on gangliosides; the initial rate of hydrolysis by neuraminidase I was 74 times, and its maximum velocity constant was 91 times those of neuraminidase II. The addition of sodium cholate markedly stimulated the enzymatic hydrolysis of gangliosides, and increased the maximum velocity constant of neuraminidase I twofold and that of neuraminidase II 143-fold. Although neuraminidases I and II were able to hydrolyze (alpha,2-3), (alpha,2-6), and (alpha,2-8) linkages, the initial rate of hydrolysis of N-acetylneuraminosyl-alpha,2-6-lactose was greater than that of the alpha,2-3-isomer.     

The linkage of sialic acid in the Ehrlich ascites-carcinoma cell surface membrane

1. The sialic acid content of fresh and fixed Ehrlich ascites cells was determined by incubation with neuraminidase and analysis of the supernatants. 2. The content of sialic acid was also determined on ultrasonically disrupted cells either with or without prior neuraminidase treatment, and the location of sialic acid in the cell is discussed. 3. The sialic acids, cleaved from cells by neuraminidase, were identified chromatographically. 4. Proteolytic enzymes were used to isolate from cells a mucopeptide containing sialic acid and galactosamine in almost equimolar proportions. 5. The nature of the carbohydrate-amino acid bond in the muco-peptide was investigated by alkaline hydrolysis. 6. A suggestion is made about the particular amino acids involved in the sugar-peptide bond.