A simple method for the determination of the O-acetyl substitution pattern of the sialic acids of colonic epithelial glycoprotein

1977 ◽  
Vol 55 (5) ◽  
pp. 493-503 ◽  
Author(s):  
P. E. Reid ◽  
C. F. A. Culling ◽  
C. W. Ramey ◽  
W. L. Dunn ◽  
M. G. Clay
Keyword(s):  
Sialic Acid ◽  
Sialic Acids ◽  
Side Chain ◽  
Substitution Pattern ◽  
Commercial Sample ◽  
Simple Method ◽  
Vibrio Cholera ◽  
Acyl Substituent ◽  
Bovine Submaxillary Mucin

A simple, rapid scheme is described for the quantitative division of the sialic acids of a glycoprotein into four classes: (a) those bearing no O-acyl substituents, (b) those with an O-acyl substituent at position C7, (c) those substituted at C9, and (d) those substituted at C8 or which are either di-(C7C8, C7C9, C8C9) or tri-(C7C8C9) substituted. This scheme can be applied to samples containing as little as 230 μg of sialic acid and requires neither acid hydrolysis nor expensive instrumentation. In combination with previously published techniques it is possible to estimate: (a) the side-chain substitution pattern of those sialic acids which are Vibrio Cholera neuraminidase (EC 3.2.1.18) insensitive, (b) the percentage of sialic acids which are Vibrio Cholera neuraminidase insensitive because of an ester substituent at either position C4 or C1, and (c) the percentage of those sialic acids which bear more than one side-chain substituent.The application of this procedure to epithelial glycoproteins isolated from the colons of man and rat and to a commercial sample of bovine submaxillary mucin is described. Evidence is presented which suggests that either these glycoproteins contain little or no C9-O-acetyl sialic acids and (or) that, under the conditions used, such acids are oxidized to completion.

Removal of O-acetylated sialic acids from rat colonic epithelial glycoproteins by cell-free extracts of rat faeces

1983 ◽  
Vol 61 (8) ◽  
pp. 868-874 ◽  
Author(s):  
H. Poon ◽  
P. E. Reid ◽  
C. W. Ramey ◽  
W. L. Dunn ◽  
M. G. Clay
Keyword(s):  
Sialic Acid ◽  
Acid Content ◽  
Minimal Medium ◽  
Wistar Rat ◽  
Sialic Acids ◽  
Side Chain ◽  
Sialic Acid Content ◽  
Vibrio Cholera ◽  
Sterile Cell

Sterile, cell-free, extracts of freshly defaecated Wistar rat faeces in a pH 7.0 "minimal medium" contain neuraminidase(s), capable of removing sialic acids both with and without side-chain substituents from bovine submandibular mucin and rat colonic epithelial glycoproteins, and an esterase which removes O-acetyl substituents from the side chain of sialic acid residues. Studies of the removal of sialic acids from bovine submandibular mucin and rat colonic epithelial glycoproteins indicated that (i) the faecal enzymes removed a greater proportion of the sialic acid of both the de-O-acetylated and native glycoproteins than was removed with Vibrio cholera neuraminidase, (ii) sialic acids were removed more rapidly from de-O-acetylated glycoproteins, and (iii) the resistance to removal of sialic acids was apparently dependent at least in part upon the O-acetyl sialic acid content of the substrate.

scholarly journals Apparent failure of 2-hydroxy-3-naphthoic acid hydrazide to block the periodic acid-Schiff reactivity of sialic acids. Implications for the PANFOPAS method.

1983 ◽  
Vol 31 (9) ◽  
pp. 1142-1144 ◽  
Author(s):  
P E Reid ◽  
C F Culling
Keyword(s):  
Sialic Acid ◽  
Periodic Acid ◽  
Acid Hydrazide ◽  
Sialic Acids ◽  
Side Chain ◽  
Substitution Pattern ◽  
Periodic Acid Schiff ◽  
Naphthoic Acid ◽  
Vicinal Diols ◽  
Anionic Groups

Histochemical studies of the mechanism of staining of the periodic acid/2,-hydroxy-3-naphthoic acid hydrazide/Fast Black B/saponification/periodic acid-Schiff (PANFOPAS) method indicate that while 2-hydroxy-3-naphthoic acid hydrazide blocks the periodate engendered Schiff reactivity of vicinal diols unassociated with anionic groups, it fails to block such reactivity in sialic acid residues. It is suggested, therefore, that the positive Schiff staining observed following application of the PANFOPAS method to colonic epithelial mucins may be due to sialic acids without side chain substituents (or which are substituted at C7 or C9) in addition to sialic acids substituted at C8 (or which are di- or trisubstituted). Consequently the PANFOPAS method cannot be used to study the side chain substitution pattern of the sialic acids of epithelial mucins.

COMPARATIVE ACTION OF VARIOUS OESTROGENIC COMPOUNDS ON MOUSE VAGINAL SIALIC ACIDS (II)

1969 ◽  
Vol 62 (4) ◽  
pp. 663-670 ◽  
Author(s):  
Lars Carlborg
Keyword(s):  
Sialic Acid ◽  
Response Curve ◽  
Sialic Acids ◽  
Clear Cut ◽  
Suitable Parameter ◽  
Sufficient Degree ◽  
Comparative Action ◽  
Relative Potencies ◽  
Good Agreement

ABSTRACT Oestrogens administered in lower doses than necessary to induce full cornification of the mouse vagina induce mucification. It was shown previously that the degree of mucification could be estimated by quantitative determination of sialic acids. A suitable parameter for oestrogen assay was the measurement of vaginal sialic acid concentration which exhibited a clear cut dose response curve. Eleven assays of various oestrogens were performed with this method. Their estimated relative potencies were in good agreement with other routine oestrogen assays. A statistically sufficient degree of precision was found. The sensitivity was of the same order, or slightly higher, than the Allen-Doisy test.

Glycoengineering the N-acyl side chain of sialic acid of human erythropoietin affects its resistance to sialidase

2012 ◽  
Vol 393 (8) ◽  
pp. 777-783 ◽  
Author(s):  
Anselm Werner ◽  
Rüdiger Horstkorte ◽  
Dagobert Glanz ◽  
Karina Biskup ◽  
Véronique Blanchard ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Culture Medium ◽  
Sialic Acids ◽  
Side Chain ◽  
Cell Culture Medium ◽  
Terminal Position ◽  
Human Erythropoietin ◽  
Recombinant Glycoproteins ◽  
Complex Glycans ◽  
Over Time

Abstract During the last years, the use of therapeutic glycoproteins has increased strikingly. Glycosylation of recombinant glycoproteins is of major importance in biotechnology, as the glycan composition of recombinant glycoproteins impacts their pharmacological properties. The terminal position of N-linked complex glycans in mammals is typically occupied by sialic acid. The presence of sialic acid is crucial for functionality and affects the half-life of glycoproteins. However, glycoproteins in the bloodstream become desialylated over time and are recognized by the asialoglycoprotein receptors via the exposed galactose and targeted for degradation. Non-natural sialic acid precursors can be used to engineer the glycosylation side chains by biochemically introducing new non-natural terminal sialic acids. Previously, we demonstrated that the physiological precursor of sialic acid (i.e., N-acetylmannosamine) can be substituted by the non-natural precursors N-propanoylmannosamine (ManNProp) or N-pentanoylmannosamine (ManNPent) by their simple application to the cell culture medium. Here, we analyzed the glycosylation of erythropoietin (EPO). By feeding cells with ManNProp or ManNPent, we were able to incorporate N-propanoyl or N-pentanoyl sialic acid in significant amounts into EPO. Using a degradation assay with sialidase, we observed a higher resistance of EPO to sialidase after incorporation of N-propanoyl or N-pentanoyl sialic acid.

scholarly journals Elucidation of the topological parameters of N-acetylneuraminic acid and some analogues involved in their interaction with the N-acetylneuraminate lyase from Clostridium perfringens

1992 ◽  
Vol 282 (2) ◽  
pp. 511-516 ◽  
Author(s):  
E Zbiral ◽  
R G Kleineidam ◽  
E Schreiner ◽  
M Hartmann ◽  
R Christian ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Oxygen Atom ◽  
Clostridium Perfringens ◽  
Pyruvic Acid ◽  
Sialic Acids ◽  
Side Chain ◽  
Equatorial Zone ◽  
Enzyme Action ◽  
Acetylneuraminic Acid ◽  
Topological Parameters

A series of neuraminic acid derivatives modified in the side chain or at C-3, C-4 or C-5 were tested as substrates of inhibitors of N-acetylneuraminate lyase (EC 4.1.3.3) from Clostridium perfringens. The results, together with Km and Ki values reported previously, indicate that the region most important for the binding of sialic acids is an equatorial zone reaching from C-8 via the ring oxygen atom to C-4 of the sugar molecule, whereas the substituents at C-9 and C-5 may be varied to a higher extent without significantly disturbing enzyme action. It is shown that stereo-electronic factors are responsible for the immediate heterolytic fragmentation of the cyclic sialic acid into pyruvic acid and 2-acetamidomannose or a related C-6 sugar.

A method for the simultaneous estimation of free and ketosidically bound sialic acids

1977 ◽  
Vol 55 (7) ◽  
pp. 778-782 ◽  
Author(s):  
C. F. A. Culling ◽  
P. E. Reid ◽  
C. W. Ramey ◽  
W. L. Dunn ◽  
M. G. Clay
Keyword(s):  
Sialic Acid ◽  
Room Temperature ◽  
Thiobarbituric Acid ◽  
Sialic Acids ◽  
Simultaneous Estimation ◽  
Sodium Metaperiodate ◽  
Acetylneuraminic Acid ◽  
Acid Reagent ◽  
Bovine Submaxillary Mucin ◽  
Free Sialic Acid

Various methods for the estimation of free and ketosidically bound sialic acid were investigated for accuracy and specificity. It was found that oxidation with 0.025 M sodium metaperiodate in pH 7.0 phosphate buffer at room temperature for 20 min provided a simple, rapid, sensitive method whereby both the free and the ketosidically bound acid present in a mixture could be quantitatively analyzed. On completion of the oxidation step, the bound sialic acid is estimated with resorcinol reagent and the free sialic acid with thiobarbituric acid reagent. Oxidation under these conditions permitted a facile analysis of mixtures of bovine submaxillary mucin and free N-acetylneuraminic acid, whereas the Warren thiobarbituric acid procedure gave an erroneous value for free sialic acid.

Evaluation of 1,10-Phenanthroline as a Reagent for Sialic Acid Determinations

1974 ◽  
Vol 20 (3) ◽  
pp. 387-388 ◽  
Author(s):  
S L Snyder ◽  
N S Mathewson ◽  
P Z Sobocinski
Keyword(s):  
Sialic Acid ◽  
Complex Formation ◽  
Sialic Acids ◽  
Previous Investigator ◽  
Acetylneuraminic Acid ◽  
Specific Reagent

Abstract Use of o-phenanthroline as a reagent for determination of sialic acids was proposed by a previous investigator. This method was based on an increase in absorbance at 307 nm that occurred when solutions of o-phenanthroline and various sialic acids were mixed. It was postulated that the increased absorbance was a result of formation of specific complexes. Using N-acetylneuraminic acid, we found no evidence for complex formation. Our results indicate that the observations of the previous investigator resulted from shifts in the pH of the medium rather than from formation of specific complexes. Therefore o-phenanthroline is not a specific reagent for sialic acids and its use is not recommended.

scholarly journals An efficient and robust HPLC method to determine the sialylation levels of human epithelial cells

2021 ◽  
Author(s):  
Hyo Jeong Kim ◽  
Stephanie Schweiker ◽  
Katie Powell ◽  
Stephan Levonis
Keyword(s):  
Sialic Acid ◽  
Cell Surface ◽  
Flow Rate ◽  
Deoxycholic Acid ◽  
Ion Pairing ◽  
Hplc Method ◽  
Sialic Acids ◽  
Reverse Phase ◽  
Simple Method ◽  
Cell Matrix

Sialyltransferase, an enzyme responsible for attaching sialic acid to the cell surface, is reported to play a key role in cancer, making sialyltransferase a potent therapeutic target in drug development for cancer. Hence, this paper aimed to develop a simple method to detect and quantify sialic acids in cancer cells. An efficient method was developed using a reverse-phase ion-pairing HPLC-UV using triisopropanolamine as the ion-pairing agent with a C18 column. Neu5Ac was successfully eluted with the retention time 6.344 min with a flow rate of 0.4 mL/min. The proposed method was validated appropriately according to the AOAC guidelines (2013). This work demonstrates that the proposed method is not only relatively simple but also cost and time effective compared to pre-existing methods to successfully determine both free and protein-bound Neu5Ac in a complex cancer cell matrix. Furthermore, by applying the proposed method, a statistically significant decrease was observed for both HeLa and HuCCT1 cell lines with the application of deoxycholic acid – a known sialyltransferase inhibitor. Hence, the proposed method may be applicable to evaluate the effectiveness of sialyltransferase inhibitors.

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

1972 ◽  
Vol 129 (3) ◽  
pp. 683-693 ◽  
Author(s):  
A. Neuberger ◽  
Wendy A. Ratcliffe
Keyword(s):  
Sialic Acid ◽  
Vibrio Cholerae ◽  
Clostridium Perfringens ◽  
Slow Rate ◽  
Alkaline Treatment ◽  
Side Chain ◽  
Hydroxyl Groups ◽  
Enzymic Hydrolysis ◽  
Hydrolysis Of

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.

ACTION OF DIETHYLSTILBOESTROL ON MOUSE VAGINAL SIALIC ACIDS (I)

1969 ◽  
Vol 62 (4) ◽  
pp. 657-662 ◽  
Author(s):  
Lars Carlborg
Keyword(s):  
Sialic Acid ◽  
Acid Concentration ◽  
Dose Response ◽  
Response Curve ◽  
Sialic Acids ◽  
Mucous Cells ◽  
Total Sialic Acid ◽  
Close Relationship ◽  
Dose Dependent

ABSTRACT Diethylstilboestrol administered in lower doses than necessary to induce full vaginal cornification in spayed mice, resulted in vaginal mucification. The thickness of the layer of mucous cells was found to be dose dependent; it was low in the controls, reached a maximum at a dose of 0.02 μg, but full cornification and absence of mucification was present at 0.05 μg. Sialic acid was present in the mucoproteins, and determination of the total amount of sialic acid per vagina showed a close relationship to the histologically evaluated degree of mucification. The ratio between total sialic acid per vagina and vaginal weight (»sialic acid concentration«) gave a dose response curve with a λ value of −0.061. The possibility of using this parameter for oestrogen assay is discussed.

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