Preliminary observations on the influence of rheumatoid alpha-1-acid glycoprotein on collagen fibril formation

This study investigates the effect of alpha(1)-acid glycoprotein (AGP) isolated from both normal and rheumatoid plasma on type II collagen fibril formation. Rheumatoid samples were obtained over 2 years from two patients with early arthritis. The glycosylation of each sample was analysed to establish any correlation with fibrillogenesis. Rheumatoid AGP displays increased fucosylation compared to normal AGP. In both patients the fucosylation dipped after 1 year, then rose again over year 2. It is proposed that year 1 corresponds to the acute phase of the disease and the onset of chronic inflammation after this time produces a subsequent increase in fucosylation. Rheumatoid AGP influences type II collagen fibrillogenesis. Native fibrils were produced but with differences in the rate and extent of fibrillogenesis depending on AGP concentration and fucosylation. Low concentrations produced a decrease in fibrillogenesis rate and fibril diameter. High concentrations produced fibrils at a rate and diameter dependent on fucosylation. Highly fucosylated AGP produced narrow fibrils slowly, whereas poorly fucosylated AGP produced thicker fibrils more quickly. We propose that differences in glycosylation (especially fucosylation) of AGP are responsible for differences in collagen fibrillogenesis and this phenomenon may contribute to the exacerbation of cartilage destruction in rheumatoid arthritis.     

A preliminary evaluation of the differences in the glycosylation of alpha-1-acid glycoprotein between individual liver diseases

During the acute phase response (APR) to tissue injury or infection, the liver is responsible for the level of mediators such as cytokines required at the site of inflammation and providing the essential components for wound healing and tissue repair. Additionally there are substantial alterations in the expression of plasma proteins of hepatic origin such as alpha-1-acid glycoprotein (AGP). The APR also results in alterations to the branching, sialylation and fucosylation of the oligosaccharide chains of AGP. This study investigated whether liver damage could be correlated with changes in AGP glycosylation in groups of patients with various liver diseases (alcoholic liver disease, hepatitis B, hepatitis C, cirrhosis). Hyperfucosylation occurred in all cases of liver disease, although the hepatitis B and C samples showed a more significant increase in comparison with the others. Additionally N-acetylgalactosamine (GalNAc) was detected in the majority of the hepatitis C samples, which was unexpected since this monosaccharide is not a usual component of the N-linked oligosaccharide chains. It was also determined by concanavalin (con) A chromatography that there is a shift towards the increased branching of the oligosaccharide chains in inflammatory liver diseases compared to normal serum.     

Identification of bifucosylated glycoforms using low‐energy CID spectra

We have used tandem mass spectrometry (MS/MS)‐based analysis of glycopeptides in order to identify the composition and structure of rare glycoforms. The results illustrate utility of low‐energy MS/MS for structure identification. We have shown the presence of bifucosylated and trifucosylated glycoforms in human α‐1‐acid glycoprotein (AGP), a major plasma glycoprotein. Fucosylation in the case of AGP always occurs on the antennae; core fucosylation was not observed.     

The Non-degradative Isolation of α1-Acid Glycoprotein from Normal and Rheumatoid Plasma1

Abstract

We have developed a method for the purification of α₁-acid glycoprotein (AGP) using procedures unlikely to damage the glycoprotein structure. This was utilised to isolate AGP from samples of normal and rheumatoid plasma. The effectiveness of the purification procedure was examined by enzymatically deglycosylating each sample of AGP, separating the released oligosaccharides by chromatography on a pellicular high pH anion-exchange (HPAE) resin at pH 13 and detecting by a pulsed electrochemical (PED) method. The analytical profile for normal AGP was consistent with those previously reported thus indicating that the purification procedure did not denature the oligosaccharide chains of AGP; there was a noticeable difference between AGP in normal and rheumatoid plasma.     

Plasma alpha 1-acid glycoprotein concentration in rats with chemical liver injury.

The influence of liver injury on the plasma concentrations of alpha 1-acid glycoprotein (AGP) and albumin was examined in several different models of chemically-induced liver injury. The plasma AGP concentration in carbon tetrachloride (CCl4), allyl alcohol, bromobenzene, acetaminophen or N-nitrosodimethylamine-induced liver injury was increased to 2-3.5 times the normal level at 24 h after the intoxication. The plasma AGP concentration was unchanged in ethionine-induced liver injury and was markedly decreased in galactosamine-induced injury. The plasma albumin concentration was significantly decreased by the damage due to galactosamine, allyl alcohol or N-nitrosodimethylamine-induced liver injury, while no influence was observed by other hepatotoxin-induced liver injury. The plasma protein binding of propranolol was also determined in relation to the plasma concentrations of AGP and albumin in all the experimental models. Propranolol binding, expressed as bound to free ratio, showed a good correlation with the AGP concentration (r = 0.940; p < 0.001), but not with the albumin concentration.     

A Rheumatoid arthritis study using Raman spectroscopy

Rheumatoid arthritis (RA) is characterized by chronic inflammation of the joints and can lead to a progressive destruction of articular cartilage and bone. In this study, the specificity and sensitivity of the RA diagnostic methods based on the receiver-operating characteristic curves for monitoring C-reactive protein (CRP) and rheumatoid factor (RF) were compared with the Raman spectroscopic diagnostic method developed in this work. Sera from 24 patients with rheumatoid arthritis and from 16 healthy individuals were analyzed to assess the biochemical composition and presence of inflammatory activity by the aforementioned methods. By comparing with the clinical results for specificity and sensitivity from the RF and CRP tests, we show that the overall results from the newly developed Raman method were significantly better, with a specificity of 96%, a sensitivity of 88%, and correctly identifying 92% of the RA and healthy individuals, while the RF test gave a specificity of 100% and a sensitivity of 54%, and the CRP test gave a specificity of 87% and a sensitivity of 58%, respectively.     

The heterogeneity of the glycosylation of alpha-1-acid glycoprotein between the sera and synovial fluid in rheumatoid arthritis

Alpha-1-acid glycoprotein (AGP) is a plasma glycoprotein produced by the liver that undergoes increased production and altered glycosylation in several physiological and pathological conditions including rheumatoid arthritis. To date, although present in the synovial fluid of rheumatoid arthritis patients, there has been no evidence for the separate extra-hepatic production of AGP. This study indicates that there could be a localized production of AGP in rheumatoid synovial fluid by demonstrating that the glycosylation patterns of AGP differed between the serum and synovial fluid in the same rheumatoid patient. Serum AGP was largely composed of fucosylated tri- and tetra-antennary oligosaccharide chains while the synovial fluid contained mainly bi-antennary chains that were fucosylated to a lesser extent. This structural heterogeneity of glycosylation resulted in functional diversity; serum but not synovial AGP is able to inhibit binding to the cell adhesion molecule E-selectin through expression of antigen sialyl Lewis X.     

The putative use of alpha-1-acid glycoprotein as a non-invasive marker of fibrosis

The acute phase response to injury or infection results in alterations in the expression of the plasma proteins produced by the liver. Many of these biomolecules are glycosylated with oligosaccharide chains covalently attached to the polypeptide backbone and the extent and composition of this glycosylation can be altered in a disease-dependent manner. Of particular interest is the observation that the acute phase glycoprotein, alpha-1-acid glycoprotein (AGP) has altered glycosylation in several physiological and pathological conditions. It is posited that changes induced in liver diseases may reflect disease severity and may therefore act as a non-invasive marker of fibrosis. This study has investigated the glycosylation of AGP in the plasma of people with varying degrees of cirrhosis and fibrosis. Hyperfucosylation was observed in all disease samples in comparison to normal plasma and was significantly increased in cirrhosis. Both sialic acid and N-acetylgalactosamine (GalNAc) were negatively associated with fibrosis. Two samples were found to express GalNAc, which as a constituent of the glycosylation of serum proteins is rare. In conclusion, fucose, sialic acid and other aspects of the glycosylation of AGP are influenced by the degree of fibrosis and as such may prove a valuable prognostic indicator of the development of cirrhosis.     

Quantitative evaluation of lectin‐reactive glycoforms of α1‐acid glycoprotein using lectin affinity capillary electrophoresis with fluorescence detection

α₁‐Acid glycoprotein (AGP) was previously shown to be a marker candidate of disease progression and prognosis of patients with malignancies by analysis of its glycoforms via lectins. Herein, affinity capillary electrophoresis of fluorescein‐labeled AGP using lectins with the aid of laser‐induced fluorescence detection was developed for quantitative evaluation of the fractional ratios of concanavalin A‐reactive or Aleuria aurantia lectin‐reactive AGP. Labeled AGP was applied at the anodic end of a fused‐silica capillary (50 μm id, 360 μm od, 27 cm long) coated with linear polyacryloyl‐β‐alanyl‐β‐alanine, and electrophoresis was carried out for about 10 min in 60 mM 3‐morpholinopropane‐1‐sulfonic acid‐NaOH buffer (pH 7.35). Addition of the lectins to the anode buffer resulted in the separation of lectin‐reactive glycoform peaks from lectin‐non‐reactive glycoform peaks. Quantification of the peak area of each group revealed that the percent of lectin‐reactive AGP is independent of a labeling ratio ranging from 0.4 to 1.5 mol fluorescein/mol AGP, i.e. the standard deviation of 0.5% for an average of 59.9% (n=3). In combination with a facile procedure for micro‐purification of AGP from serum, the present procedure, marking the reactivity of AGP with lectins, should be useful in determining the prognosis for a large number of patients with malignancies.     

CIEF with hydrodynamic and chemical mobilization for the separation of forms of alpha-1-acid glycoprotein

Alpha-1-acid glycoprotein (AGP) is a protein that exists in different forms, which is due to variations in the amino acid sequence and/or in the glycosidic part of the protein. These differences confer to these forms, among other characteristics, diverse pIs. Changes in these forms of AGP have been correlated to modifications of the pathophysiological conditions of the individuals. One of the analytical techniques employed for their study has been IEF performed in slab gels. CIEF method with hydrodynamic and chemical mobilization, involving an isotachophoretic process, is developed in this work to separate up to 12 bands of forms of standard AGP, which is proposed as a more reproducible, quantitative, less sample-consuming, and more automated one than conventional IEF. The challenge of this work has been the development of a CIEF method for the separation of bands of a very acidic protein (pI range: 1.8-3.8) in a capillary. Intraday RSD values < or = 1.7% have been achieved for the relative migration time of the AGP bands to that of an internal standard. For intraday area precision, RSD (%) in the range of 2.70-22.71% for AGP zones accounting for more than 10% of total area of AGP sample has been obtained. As a proof of the potential of the methodology proposed, an AGP sample purified from a pool of sera of patients suffering from ovary cancer is analyzed by CIEF.     

A preliminary evaluation of the functional significance of alpha-1-acid glycoprotein glycosylation on wound healing

The laying down of collagen and fibrous tissue is a key process in wound healing, however excessive collagen (and glycoprotein) deposition causes hypertrophic and keloid scars, eg after burns. Collagen synthesis is increased in these scars compared with normal healing, as is collagenase activity, which controls the degradation pathway of collagen. The processes of wound healing are inextricably linked to those of the acute-phase response (APR): alpha-1-acid glycoprotein (AGP), a plasma glycoprotein that undergoes both an increase in concentration and an alteration in its glycosylation pattern during the APR. This study determined that AGP isolated from the plasma of burns patients was of an increased concentration and altered glycosylation pattern compared with normal plasma and was capable of directly interacting with type I collagen. It also had a profound effect on both collagen fibril formation and collagenase activity, to a degree dependent upon the percentage body surface area burned. Additionally, the results obtained provided the basis for predicting the formation of hypertrophic scars.     

Biointeraction analysis of carbamazepine binding to α1‐acid glycoprotein by high‐performance affinity chromatography

Interactions of the drug carbamazepine with the serum protein α₁‐acid glycoprotein (AGP) were examined by high‐performance affinity chromatography. Frontal analysis studies with an immobilized AGP column and control column indicated carbamazepine had both low‐affinity interactions with the support and high‐affinity interactions with AGP. When a correction was made for binding to the support, the association equilibrium constant measured at pH 7.4 and 37°C for carbamazepine with AGP was 1.0 (±0.1)×10⁵ M^?1, with values that ranged from 5.1 to 0.58×10⁵ M^?1 in going from 5 to 45°C. It was found in competition studies that these interactions were occurring at the same site that binds propranolol on AGP. Temperature studies indicated that the change in enthalpy was the main driving force for the binding of carbamazepine to AGP. These results provide a more complete picture of how carbamazepine binds to AGP in serum. This report also illustrates how high‐performance affinity chromatography can be used to examine biological interactions and drug–protein binding in situations in which significant interactions for an analyte are present with both the chromatographic support and an immobilized ligand.     

Investigating the binding measurements of human α-acid glycoprotein with chlorambucil and dacarbazine in the presence of imidazolium based -ionic liquid by affinity capillary electrophoresis

Human alpha (α1)-acid glycoprotein (AGP) is an acute phase protein whose plasma concentration increases several-folds in the presence of various diseases. The variability in AGP plasma concentration is expected to have a huge impact on the drug binding equilibrium. Therefore, a precise measurement of AGP-drug binding is of great demand for drug development. In the current study, an ionic liquid-based aqueous two-phase system combined with affinity capillary electrophoresis (ILATPS/ACE) was utilised in order to improve the accuracy of AGP-drug binding analysis through the measurements of electrophoretic mobilities. The utilisation of ILATPS has shown to have a positive impact on the stability of AGP activity solution during the storage for an extended period of time. In addition, the effect of various alkyl chains (C2-C10) of imidazolium-based ILs with concentrations ranging between 10.00 and 1000.0 μmol L⁻¹ on the AGP binding with the anti-cancer drugs chlorambucil (CHL) and dacarbazine (DAC) was examined by the system developed (ILATPS/ACE). A 100.00 μmol L⁻¹ 1-ethyl-3-methylimidazolium chloride (EMImCl) prepared in the physiological buffer conditions containing AGP (5.00–100.00 µmol L⁻¹) has provided an accurate apparent binding constant of 1.99 ± 0.11 and 6.95 ± 0.14 L mmol⁻¹ with CHL and DAC respectively. Apart from the ACE analysis, EMImCl/phosphate buffer solution was found to be a distinguished system that could lengthen the stability of AGP activity for a period of time reaching 90 days during the solution storage at 4.00 °C. This effect is thought to be due to the easy conversion of one-phase EMImCl/phosphate buffer/AGP at the ambient lab temperature into the two-phase solution at refrigerator temperature, 4.00 °C, and vice versa. Therefore, the ILATP/ACE system could be used to enhance the accuracy for other AGP-drug bindings with a fast, easy to use, and cost-effective analysis.     

Rational strategy of magnetic relaxation switches for glycoprotein sensing

A rational strategy of magnetic relaxation switches was proposed here to detect a(1)-acid glycoprotein (AGP), an acute phase a-globulin plasma glycoprotein. The assay was based on the relaxation time change between the aggregation of magnetic nanoparticles with concanavalin A and the redispersion with AGP, which can avoid the prozone effect and improve the detection accuracy. The assay was an easy and efficient method with two mixing steps and one measurement step, showing a detection limit of 0.66 nM in 0~0.3 μg mL(-1) AGP, which was far lower than its normal level in human plasma.     

Characterization of basic drug-human serum protein interactions by capillary electrophoresis

Drug-protein interactions are determining factors in the therapeutic, pharmacodynamic and toxicological drug properties. The affinity of drugs towards plasmatic proteins is apparently well established in bibliography. Albumin (HSA) especially binds neutral and negatively charged compounds; alpha(1)-acid glycoprotein (AGP) binds many cationic drugs, lipoproteins bind to nonionic and lipophilic drugs and some anionic drugs while globulins interact inappreciably with the majority of drugs. In this paper, the characterization of the interaction between cationic drugs, beta-blockers and phenotiazines towards HSA, AGP, and both HSA + AGP mixtures of proteins under physiological conditions by CE-frontal analysis is presented. Furthermore, the binding of these drugs to all plasmatic proteins is evaluated by using ultrafiltration and CE. The results indicate that the hydrophobic character of compounds seems to be the key factor on the interaction between cationic drugs towards proteins. In fact, hydrophobic basic drugs bind in great extension to HSA, while hydrophilic basic drugs present low interactions with proteins and bind especially to AGP.     

One-step purification of rat plasma alpha 1-acid glycoprotein by antibody affinity chromatography: application to normal and inflamed rat sera

Most purification procedures used previously to isolate alpha 1-acid glycoprotein (AGP) from plasma can lead to some alterations in its carbohydrate moiety. An immunoaffinity chromatographic method is proposed for purifying in one step rat plasma AGP without any detectable modification of its glycan moiety. Crossed immunoaffinoelectrophoresis with concanavalin A before and after purification showed identical patterns, suggesting no glycan selection during the purification. In the same way no desialylation occurred during the purification step. This immunoaffinity chromatographic procedure provided evidence of a decreased level of fucosyl residues in turpentine oil rat plasma AGP compared with normal rat plasma AGP.     

Statistical evaluation of CZE-UV and CZE-ESI-MS data of intact α-1-acid glycoprotein isoforms for their use as potential biomarkers in bladder cancer

α-1-acid glycoprotein (AGP) is a highly heterogeneous protein that presents a vast number of isoforms (molecules of the protein differing in its peptidic and/or glycosidic moieties). In the last years, several authors have studied the potential use of AGP as a cancer biomarker. These studies focus on the correlation of different features of AGP structure (i.e. fucosylation, antennarity) with cancer or on the total protein blood concentration. In this study, the potential of CZE-UV and CZE-ESI-MS analysis of intact AGP isoforms to study the correlation of this protein with bladder cancer is shown. Samples from 16 individuals (eight healthy, eight bladder cancer) were analyzed and characterized in great detail including data on intact protein isoforms and on released glycans. The analytical data were evaluated employing different statistical techniques (ANOVA; principal component analysis, PCA; linear discriminant analysis; and partial least squares-discriminant analysis). Statistical differences between the two groups of study were observed. The best results were obtained by linear discriminant analysis of the CZE-ESI-MS data for intact AGP isoforms (93.75% of correct classification). Due to MS characterization, it can be observed that differences between the samples are mainly due to higher abundance of AGP isoforms containing tri- and tetra-antennary fucosylated oligosaccharides in cancer patients. The results show the great potential of CE-MS in combination with advanced data processing for the use of intact protein isoforms as disease biomarkers.     

Investigation of genetic variants of α-1 acid glycoprotein by ultra-performance liquid chromatography–mass spectrometry

Genetic variants of human plasma alpha-1 acid glycoprotein (AGP) have been studied in cancer, compared with a group of healthy control. AGP has four genetic variants: AGP F1, F2, and S variants correspond to the ORM1 gene whereas AGP A corresponds to the ORM2 gene. The proportion of ORM1 and ORM2 variants were studied in plasma using a novel UPLC–MS method. Plasma total AGP level was 0.5 ± 0.2 g L⁻¹ and the proportions of the ORM1 and ORM2 variants were 76.3 ± 8.2% and 23.7 ± 8.2%, respectively. In cancer plasma AGP levels increased fourfold and the proportion of ORM1 variants increased to 88.7 ± 6.8%. Changes in the proportion of genetic variants due to cancer were clearly significant, as shown by statistical analysis. Three different cancer types have been studied, lymphoma, melanoma, and ovarian cancer. The results did not show any difference depending on cancer type. The results indicate that, in accordance with prior expectations, the ORM1 variant is predominantly responsible for the acute-phase property of AGP.     

Protective Effect of Withania somnifera and Cardiospermum halicacabum Extracts Against Collagenolytic Degradation of Collagen

The irreversible destruction of extracellular matrix (ECM) such as cartilage, tendon, and bone that comprise synovial joints is the hallmark of both rheumatoid arthritis and osteoarthritis by over-expression of matrix metalloproteinase (MMP)-collagenases. We report herein the detailed study on the inhibitory effects of Withania somnifera extract (WSE) and Cardiospermum halicacabum extract (CHE) on Clostridium histolyticum collagenase (ChC) activity against the degradation of the ECM component of bovine Achilles tendon type I collagen by hydroxyproline assay method. Interaction of WSE and CHE with ChC exhibited 71% and 88% inhibition, respectively, to the collagenolytic activity of ChC against collagen degradation, and the inhibition was found to be concentration-dependent. The inhibition kinetics of ChC by both the extracts has been deduced from the extent of hydrolysis of N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala. Both WSE and CHE are provided competitive and mixed type inhibition on ChC activity, respectively. Circular dichroism studies of ChC on treatment with WSE and CHE revealed changes in the secondary structure of collagenase. These results suggest that the WSE and CHE facilitated collagen stabilization through collagenase inhibition.