The carbohydrate units of human plasma α1-Acid glycoprotein

doi:10.1016/0005-2795(77)90080-0

Biochimica et Biophysica Acta (BBA) - Protein Structure

Volume 492, Issue 2, 24 June 1977, Pages 291-302

https://doi.org/10.1016/0005-2795(77)90080-0 Get rights and content

Abstract

α₁-Acid glycoprotein derived from pooled normal human plasma was desialyzed, reduced and carboxymethylated and then digested with chymotrypsin. The resulting glycopeptide mixture, separated from the peptides by gel filtration through Sephadex G-25, was subsequently fractionated by chromatography on CM- and DEAE-celluloses and Dowex 50. The five glycopeptides which were obtained were homogeneous with regard to their electrophoretic properties, amino-terminal amino acids and amino acid compositions, were found to be derived from five specific regions of the polypeptide chain. Each glycopeptide was then subjected to exhaustive treatment with pronase and chromatographed on Dowex-50 at a low ionic strength. Each chromatogram yielded a group of five to eight pronase-glycopeptides, and a total of 26 homogeneous carbohydrate-carrying peptides was obtained. The carbohydrate composition of these compounds indicated that (a) fucose was associated with only the minor glycopeptides of each group of compounds, (b) the heteropolysaccharide units that are linked to each of the five glycosylation sites of the protein are different in size (c) as judged by the composition of the heteroglycans approximately 60% of the carbohydrate units have compositions similar to those of other typical plasma glycoproteins while the remainder of the carbohydrate moiety of this α-globulin is characterized by unusually high galactose contents and high galactose-mannose ratios. Of equal importance, this paper describes a procedure for the isolation and purification of the heteroglycans of each of the carbohydrate attachment sites of a glycoprotein which possesses multiglycosylation sites.

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Fucosylated AGP glycopeptides as biomarkers of HNF1A-Maturity onset diabetes of the young
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We previously demonstrated that antennary fucosylated N-glycans on plasma proteins are regulated by HNF1A and can identify cases of Maturity-Onset Diabetes of the Young caused by HNF1A variants (HNF1A-MODY). Based on literature data, we further postulated that N-glycans with best diagnostic value mostly originate from alpha-1-acid glycoprotein (AGP). In this study we analyzed fucosylation of AGP in subjects with HNF1A-MODY and other types of diabetes aiming to evaluate its diagnostic potential.
A recently developed LC-MS method for AGP N-glycopeptide analysis was utilized in two independent cohorts: a) 466 subjects with different diabetes subtypes to test the fucosylation differences, b) 98 selected individuals to test the discriminative potential for pathogenic HNF1A variants.
Our results showed significant reduction in AGP fucosylation associated to HNF1A-MODY when compared to other diabetes subtypes. Additionally, ROC curve analysis confirmed significant discriminatory potential of individual fucosylated AGP glycopeptides, where the best performing glycopeptide had an AUC of 0.94 (95% CI 0.90–0.99).
A glycopeptide based diagnostic tool would be beneficial for patient stratification by providing information about the functionality of HNF1A. It could assist the interpretation of DNA sequencing results and be a useful addition to the differential diagnostic process.
Silica embedded carbon nanosheets derived from biomass acorn cupule for non-enzymatic, label-free, and wide range detection of α <inf>1</inf>-acid glycoprotein in biofluids
2021, Analytica Chimica Acta
In this work, we demonstrate the first report on a low-cost, non-enzymatic, label-free electrochemical sensing of alpha-1-acid-glycoprotein (α₁GP) biomarker in biofluids using silica embedded carbon nanosheets (SEC) derived from acorn cupules biomass. The SEC/GCE sensor exhibits low detection limit (LOD) of 30 ng/mL (LOD = 3 s/m) which is far below the lowest physiological concentration of α₁GP and a wide linear detection range from 100 ng/mL to 10 mg/mL which covers entire clinical concentration range reported for various diseases like cancer, sepsis, etc. The sensing mechanism of the sensor relies on the direct electrooxidation of sialic acid from the α₁GP structure which hinder the interfacial electron-transfer process of [Fe (CN)₆]^3-/4- redox probe at the electrode-electrolyte interface. The sensor exhibits excellent selectivity and stability towards detection of α₁GP which is ascribed to the presence of embedded silica nanoparticles on the surface of the carbon nanosheets. The successful determination of α₁GP in the simulated blood serum sample with good recovery percentage (i.e., from ∼94.03% to ∼103.50%) proves the feasibility of the sensor towards clinical applications. The overall efficacy of the sensor proves it as a promising low-cost, sustainable, and non-enzymatic platform for a wide variety of bioanalytical applications.
High-Throughput and Site-Specific N-Glycosylation Analysis of Human Alpha-1-Acid Glycoprotein Offers a Great Potential for New Biomarker Discovery
2021, Molecular and Cellular Proteomics
Alpha-1-acid glycoprotein (AGP) is an acute phase glycoprotein in blood, which is primarily synthetized in the liver and whose biological role is not completely understood. It consists of 45% carbohydrates that are present in the form of five N-linked complex glycans. AGP N-glycosylation was shown to be changed in many different diseases, and some changes appear to be disease-specific; thus, it has a great diagnostic and prognostic potential. However, AGP glycosylation was mainly analyzed in small cohorts and without detailed site-specific glycan information. Here, we developed a cost-effective method for a high-throughput and site-specific N-glycosylation LC-MS analysis of AGP which can be applied on large cohorts, aid in search for novel disease biomarkers, and enable better understanding of AGP’s role and function in health and disease. The method does not require isolation of AGP with antibodies and affinity chromatography, but AGP is enriched by acid precipitation from 5 μl of bloodplasma in a 96-well format. After trypsinization, AGP glycopeptides are purified using a hydrophilic interaction chromatography-based solid-phase extraction and analyzed by reversed-phase-liquid chromatography-electrospray ionization-MS. We used our method to show for the first time that AGP N-glycan profile is stable in healthy individuals (14 individuals in three time points), which is a requirement for evaluation of its diagnostic potential. Furthermore, we tested our method on a population including individuals with registered hyperglycemia in critical illness (59 cases and 49 controls), which represents a significantly increased risk of developing type 2 diabetes. Individuals at higher risk of diabetes presented increased N-glycan branching on AGP’s second glycosylation site and lower sialylation of N-glycans on AGP’s third and AGP1’s fourth glycosylation site. Although this should be confirmed on a larger prospective cohort, it indicates that site-specific AGP N-glycan profile could help distinguish individuals who are at risk of type 2 diabetes.
Study of the binding affinity between imatinib and α-1 glycoprotein using nuclear spin relaxation and isothermal titration calorimetry
2020, International Journal of Biological Macromolecules
Imatinib is a selective tyrosine kinase inhibitor, successfully used for the treatment of chronic myelogenous leukaemia and gastrointestinal stromal tumors. Binding of drugs to proteins influence their pharmacokinetic and pharmacodynamics action. In the blood, the drug is distributed in the body in the free form or bound to plasma protein. Albumin and α-1 glycoprotein (AGP) are plasma proteins with the highest affinity for drug substances. Drugs which are weak acids mainly bind to plasma albumin, while drugs that are bases have affinity for α-1 glycoprotein. The main goal of this study is to quantitatively evaluate the interaction between imatinib mesylate (IMT) and α-1 glycoprotein to characterize the nature and forces underlying the formation of a molecular complex. Relaxation experiments provide quantitative information about the relationship between the binding affinity and structure of IMT. Thus, association constant was determined as K_a = 873.36 M⁻¹. The ITC data revealed that the binding was an entropy driven process and the association constant K_a = 3.22 × 10³ M⁻¹, with a 1:1 stoichiometry. The results obtained by NMR and ITC were complemented with a molecular docking study.
A fast and convenient solid phase preparation method for releasing N-glycans from glycoproteins using trypsin- and peptide-N-glycosidase F (PNGase F)-impregnated polyacrylamide gels fabricated in a pipette tip
2020, Journal of Pharmaceutical and Biomedical Analysis
An efficient deglycosylation process is a key requirement for the identification and characterization of glycosylation during the production and purification of therapeutic antibodies. PNGase F is widely used for the deglycosylation of N-linked glycans. The commonly-used in-solution deglycosylation method is relatively time-consuming and requires several hours up to overnight for complete removal of all N-linked glycans. In order to develop a simple and efficient method for the rapid release of N-linked glycans from glycoproteins, we fabricated trypsin- and PNGase F-impregnated polyacrylamide gels in a commercial 200 μL volume pipette tip. Our enzyme reactor is based on simple photochemical copolymerization of monomers using the following procedure: (1) a pipette tip was filled with a gel solution comprising acrylamide, N,N’-methylene-bis-acrylamide containing PNGase F or trypsin with 2,2-azobis(2-methyl-N-(2-hydroxyethyl) propionamide) as a photocatalytic initiator; and (2) in situ polymerization of gel solution approximately 30 mm from the tip was performed by irradiation with a 365 nm blue LED beam from a distance 10 mm. The fixed enzymes maintained their activities in the polyacrylamide gel and the reaction was completed by 40 iterations of suction and discharge with a pipette (hereafter referred to as manual pipetting times) for 8 min with each enzyme digestion. Capillary electrophoresis (CE) of released glycans labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS) demonstrated quantitative recovery of glycans from selected glycoproteins.
An overview of albumin and alpha-1-acid glycoprotein main characteristics: highlighting the roles of amino acids in binding kinetics and molecular interactions
2019, Heliyon
Citation Excerpt :
The AGP protein consists of about 59% peptide residues and 41% carbohydrates - of which about 11% are sialic acids (or 12% according to Hochepied et al. (2003)). This high sialic content contributes to the negative charge on its surface (Kremer et al., 1988; Schmid et al., 1977) and determines the nature of interactions that this glycoprotein can have with biological membranes. There are five structurally heterogeneous carbohydrate entities (five N-glycans) with mono-, bi-, tri-, and tetra-antennary glycans covalently linked to five asparagine (Asn) residues, which form N-glycosylation sites (Asn-15, Asn-38, Asn-54, Asn-75, Asn-85) to the most active gene ORM1 (variant F1*S) between the two main genes of AGP (i.e. ORM1 and ORM2) (Dente et al., 1987; Fournier et al., 2000; Taguchi et al., 2013; Yoshima et al., 1981; Yuasa et al., 1997).
Although Albumin (ALB) and alpha-1-acid glycoprotein (AGP) have distinctive structural and functional characteristics, they both play a key role in binding a large variety of endogenous and exogenous ligands.
An extensive binding to these plasma proteins could have a potential impact on drugs disposition (e.g. bioavailability, distribution and clearance), on their innocuity and their efficacy. This review summarizes the common knowledge about the structural and molecular characteristics of both ALB and AGP in humans, and about the most involved amino acids in their high-affinity binding pockets. However, the variability in residues found in binding pockets, for the same species, allows each plasma protein to interact differently with the ligands. The protein-ligand interaction influences differently the disposition of drugs that bind to either of these plasma proteins.
The content of this review is useful for the design of new drug entities with high-binding characteristics, in qualitative and quantitative modelling (e.g. in vitro-in vivo extrapolations, 3D molecular docking, interspecies extrapolations), and for other interdisciplinary research.

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The carbohydrate units of human plasma α1-Acid glycoprotein

Abstract

J. Biol. Chem.

Biochem. Biophys. Acta (1962)

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Acta

Biochem. Biophys. Acta

J. Chromatogr.

J. Biol. Chem.

Biochemistry

Biochemistry

Biochem. J.

The carbohydrate units of human plasma α₁-Acid glycoprotein