Analysis of N-acetyl-4-O-acetylneuraminic-acid-containing N-linked carbohydrate chains released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Application to the structure determination of the carbohydrate chains of equine fibrinogen.
Abstract: The carbohydrate chains of equine fibrinogen were enzymatically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. The oligosaccharides obtained were fractionated by a combination of FPLC and HPLC and analyzed by 500-MHz 1H-NMR spectroscopy. Four monosialo and four disialo diantennary N-acetyllactosamine type of carbohydrate chains occur: (formula; see text)
Publication Date: 1989-03-01 PubMed ID: 2707257DOI: 10.1111/j.1432-1033.1989.tb14620.xGoogle Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The study is about the structural analysis of the carbohydrate chains of equine fibrinogen using the enzyme peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, with the help of fractionation techniques like fast protein liquid chromatography (FPLC) and high performance liquid chromatography (HPLC), along with 500-MHz 1H-NMR spectroscopy.
Research Methodology
- The researchers initiated the process by obtaining the carbohydrate chains of equine fibrinogen with the help of the enzyme peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Equine fibrinogen is a protein derived from horses that assists in the clotting of blood.
- These obtained carbohydrate chains, also known as oligosaccharides, were then subjected to separation techniques using FPLC and HPLC. These techniques fractionate, or separate, the complex mixture into its constituent molecules for individual study.
- FPLC is a form of column chromatography used to separate or purify proteins from complex mixtures. HPLC, in contrast, is a type of column chromatography that is utilized to identify, quantify and separate each component in a mixture.
Results and Findings
- Post-fractionation, the researchers used 500-MHz 1H-NMR spectroscopy for the analysis of the oligosaccharides. Spectroscopy is a technique used to study the interaction between matter and radiated energy, here they used the 1H-NMR variation which investigates the hydrogen-1 nuclei within the molecules.
- The in-depth analysis divulged the presence of four monosialo and four disialo diantennary N-acetyllactosamine type of carbohydrate chains. These carbohydrate chains are part of glycoconjugates, which play functional roles in cell-cell interactions and recognition.
- The researchers presented the detailed formula that depicts the structural organization of the discovered carbohydrate chains.
Significance of the Study
- The research serves as an important step forward in understanding the structure of the carbohydrate chains of equine fibrinogen.
- The findings could be instrumental in future studies aimed at analyzing similar protein structures. Furthermore, the well-documented process could assist in the replication of the study for other proteins with carbohydrate chains.
Cite This Article
APA
Damm JB, Voshol H, Hård K, Kamerling JP, Vliegenthart JF.
(1989).
Analysis of N-acetyl-4-O-acetylneuraminic-acid-containing N-linked carbohydrate chains released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Application to the structure determination of the carbohydrate chains of equine fibrinogen.
Eur J Biochem, 180(1), 101-110.
https://doi.org/10.1111/j.1432-1033.1989.tb14620.x Publication
Researcher Affiliations
- Department of Bio-Organic Chemistry, Utrecht University, The Netherlands.
MeSH Terms
- Acetylation
- Amidohydrolases
- Animals
- Carbohydrate Conformation
- Carbohydrates / analysis
- Chromatography, High Pressure Liquid
- Chromatography, Ion Exchange
- Fibrinogen / analysis
- Horses
- Magnetic Resonance Spectroscopy
- Molecular Structure
- Oligosaccharides / analysis
- Peptide Fragments / analysis
- Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
- Sialic Acids / analysis
Citations
This article has been cited 7 times.- Schauer R, Kamerling JP. Exploration of the Sialic Acid World.. Adv Carbohydr Chem Biochem 2018;75:1-213.
- Vliegenthart JF. The complexity of glycoprotein-derived glycans.. Proc Jpn Acad Ser B Phys Biol Sci 2017;93(2):64-86.
- Di Patrizi L, Rosati F, Guerranti R, Pagani R, Gerwig GJ, Kamerling JP. Structural characterization of the N-glycans of gpMuc from Mucuna pruriens seeds.. Glycoconj J 2006 Nov;23(7-8):599-609.
- Navazio L, Baldan B, Mariani P, Gerwig GJ, Vliegenthart JF. Primary structure of the N-linked carbohydrate chains of Calreticulin from spinach leaves.. Glycoconj J 1996 Dec;13(6):977-83.
- Hokke CH, van der Ven JG, Kamerling JP, Vliegenthart JF. Action of rat liver Gal beta 1-4GlcNAc alpha(2-6)-sialyltransferase on Man beta 1-4GlcNAc beta-OMe, GalNAc beta 1-4GlcNAc beta-OMe, Glc beta 1-4GlcNAc beta-OMe and GlcNAc beta 1-4GlcNAc beta-OMe as synthetic substrates.. Glycoconj J 1993 Feb;10(1):82-90.
- L'Hôte C, Berger S, Bourgerie S, Duval-Iflah Y, Julien R, Karamanos Y. Use of porcine fibrinogen as a model glycoprotein to study the binding specificity of the three variants of K88 lectin.. Infect Immun 1995 May;63(5):1927-32.
- Hård K, Mekking A, Kamerling JP, Dacremont GA, Vliegenthart JF. Different oligosaccharides accumulate in the brain and urine of a cat with alpha-mannosidosis: structure determination of five brain-derived and seventeen urinary oligosaccharides.. Glycoconj J 1991 Feb;8(1):17-28.
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