Non enzymatic glycosylation of alpha-1-proteinase inhibitor of human plasma.M Phadke, FR Billimoria, V Ninjoor
Department of Biochemistry, L.T.M. Medical College, Sion, Mumbai.
Human plasma contains inhibitors, which control the activity of proteolytic enzymes. Alpha-1-proteinase inhibitor and alpha-2-macroglobulin are two of them present in high concentration in human plasma, which inhibit action of trypsin among other proteinases. The trypsin inhibitory capacity (TIC) of human plasma is observed to be decreased in pathological conditions like diabetes mellitus. The mechanisms of decrease in TIC was due to nonenzymatic glycosylation of alpha-1-proteinase inhibitor (A1PI). A1PI was partially purified from normal human plasma by steps involving ammonium sulphate precipitation, DEAE Sepharose CL6B chromatography, Concanavalin A Sepharose Chromatography and Sephadex G-100 Gel filtration. Purified inhibitor was glycosylated in vitro by incubating it with varying glucose concentrations, under nitrogen for different periods of time in reducing conditions. After glycosylation, the molecular weight of inhibitor increased from 52 kDa to 57 KDa because of binding with glucose molecules. The percent free amino groups in the protein decreased with increasing glucose concentration and days of incubation. The TIC of such modified inhibitor decreased significantly. Decrease in TIC was dependent on the glucose concentration and period of incubation used during in-vitro glycosylation of native inhibitor.
Keywords: Chromatography, Diabetes Mellitus, metabolism,Glycosylation, Human, Serine Proteinase Inhibitors, blood,isolation &purification,metabolism,Trypsin Inhibitors, blood,alpha 1-Antitrypsin, isolation &purification,metabolism,
Human body fluids and tissues contain many proteinase inhibitors, which regulate the proteolytic action of various proteinases and protect the tissues from unwanted protein degradation. The activity of proteolytic enzymes or proteinases is expressed at specific locations and such activity is reduced whenever necessary. Human plasma contains many proteinase inhibitors that control the proteolytic activity of different exogenous and endogenous proteinases. Alpha-1-proteinase inhibitor (or Alpha-1-antitrypsin or A1PI) and alpha-2-macroglobulin (A2M) are the two among them, present in high concentrations. Diabetes mellitus is characterized by absolute or relative insulin deficiency leading to hyperglycemic condition. The trypsin inhibitory capacity of A2M has been reported to be decreased in condition of diabetes mellitus. However, the status of A1PI in this condition remains obscure.
Glycosylation as a non-enzymatic unregulated process is observed to be taking place in condition of hyperglycemia. Accelerated non-enzymatic glycosylation of many body proteins altering their function has been evidenced in diabetes mellitus.
The present work was undertaken to elucidate the mechanism of reduced inhibitory activity of plasma as regards A1PI in diabetes. To achieve this, A1PI was purified from normal human plasma; it was glycosylated non-enzymatically in-vitro and its inhibitory profile studied.
The alpha-1-proteinase inhibitor was purified from human plasma by method modified from Travis & Johnson (1980).
DEAE Sepharose CL6B, Concanavalin A Sepharose 4B, Sephadex G-100 and molecular weight markers were from Pharmcia Fine Chemicals, Sweden. Bovine serum albumin (BSA), trypsin and Benzoyl arginine-P-nitro aniline (BAPNA) were purchased from Sigma Chemical Co., St. Louis, U.S.A. Trinitro Benzene sulfonic acid (TNBS) was the product of Aldrich chemical Co., U.S.A.
Blood was collected from normal healthy blood donors and was checked to be free from hepatitis B virus and antibodies for Human Immunodeficiency Virus by ELISA method. Red cells separated by cold centrifugation were supplied to patients with thalassemia and the clear plasma was used for purification of the inhibitor. All steps of purification were carried out at 0-4?C unless otherwise specified.
3. Ammonium sulphate precipitation
The clear plasma was subjected to 50-80% ammonium sulphate fractionation and the precipitated protein was collected by centrifugation at 12,000 g. for 20 minutes. The precipitate was dissolved in 30mM phosphate buffer pH 6.5 and dialysed exhaustively against the same buffer to remove the salt. The dialysate was clarified by spinning at 12,000 g for 15 minutes and the insoluble material discarded.
4. DEAE Sepharose CL6B chromatography
The clear supernatant obtained from ammonium sulphate precipitation was applied on a DEAE Sepharose CL6B column (2.6 X 33 cm.) pre-equilibrated with 30mM phosphate buffer pH 6.5. The column bed was washed with the same buffer until the elute showed an absorbance at 280 nm nearing OA linear gradient of 0-200 mM Sodium chloride in 30mM Sodium phosphate buffer pH 6.5 was applied to develop the chromatogram. Fractions of 5 ml were collected with a flow rate of 20 ml/h. The fractions showing inhibitory activity were pooled and concentrated in an Amicon Diaflow cell (Amicon Corporation, Denver, U.S.A.) using YM-10 membrane.
5. Concanavalin A Sepharose chromatography
The concentrated sample from DEAE Sepharose CL6B was dialyzed against 10 mM Tris/HCL buffer pH 7.5 containing normal saline and applied on Concanavalin A Sepharose column (2.6 X 33 cm) previously equilibrated with the same buffer (buffer A). Unbound protein was eluted with Buffer A and to achieve elution of A1PI a linear gradient of 100 mM alpha-methyl-D-glucoside in buffer A was applied. The flow was maintained at rate of 10 ml/h and 2.5 ml fractions were collected. Fractions showing inhibitory activity towards trypsin were pooled and dialyzed against 50 mM Tris – HCL pH 8.0 containing 50 mM NaCl.
6. Gel filtration on Sephadex g-100
The dialysed sample from Concanavalin A Sepharose chromatography was applied on Sephadex G-100 column (2.6 X 60 cm) which was equilibrated with 50 mM Tris - HCL buffer pH 8.0 containing 50 mM NaCl. Elution was carried out with the same buffer at a flow rate of 25 ml/h and 6.2 ml fractions were collected. Active fractions were pooled and concentrated.
7. Determination of molecular weight
This was achieved on HPLC using gel permeation column TSK G 3000 SW of Pharmtia LKB. Calibration kit with BSA (67 kDa), Ovalbumin (43 kDa), Carbonic anhydrase (30 kDa). Myoglobin (17 kDa) and Cytochrome C (12.2 kDa) was used to calibrate the column. Blue dextran was employed to monitor the void volume.
8. Assay for the activity of inhibitor
Inhibitory action against proteolytic enzyme trypsin was assayed using BAPNA as the substrate. The buffer used was 50 mM Tris - HCL pH 8.0 containing 50 mM CaCl2.2H2O. Positive control was run in absence of inhibitor containing only trypsin. Extent of trypsin inhibition in presence of inhibitor was expressed as percent inhibition of trypsin by comparing with positive control.
9. Glycosylation of purified inhibitor
The partially purified inhibitor was glycosylated non-enzymatically in-vitro to assess the effect of glycosylation on the inhibitory properties. The procedure followed was based on the one used by Duell et al.
Reagents: 200 mM phosphate buffered saline (PBS) pH 7.3, 50 mM Tris - HCl buffer pH 8.0.
Method: Glycosylation was allowed to take place non-enzymatically under reducing condition created by the use of sodium-borohydride.
Purified A1PI sample, 0.5 ml corresponding to 0.59 mg was taken in each vial in a total 2 ml of 50 mM Tris - HCl buffer pH 8.0 with 12 mg/ml of sodium-borohydride. D-glucose at different concentration of 200, 300 and 500 mM was added in different sets, sealed under nitrogen and incubated at 37?C for a period of either 5 or 10 days.
Bovine serum albumin (BSA) was glycosylated simultaneously for comparing the extent of modification taking place with that of A1PI under similar experimental conditions.
Five mg BSA in 2 ml of PBS with 12 mg/ml sodium borohydride was taken in each vial. D-glucose at different concentrations of 100, 200, 300 and 500 mM was added in different sets and the vials after sealing under nitrogen were incubated at 37?C for 3, 5, 8 or 10 days.
After the incubation period, the glycosylated solution was dialyzed extensively against the same buffer in use to remove unreacted glucose and sodium-borohydride. The extent of glycosylation achieved was determined by assay with picryl sulfonic acid or trinitrobenzene sulfonic acid (TNBS).
10. Assay with TNBS
This is a sensitive method to determine the free amino groups in proteins. The optical density (OD) of unmodified protein represented 100% free amino groups in protein and from the decrease in OD after glycosylation, percent glycosylated amino groups was calculated.
Human plasma alpha-1-proteinase inhibitor was purified by ammonium sulphate precipitation, DEAE Sephadex CL6B chromatography, Con A Sepharose chromatography followed by Sephadex G-100 gel filtration.
[Table - 1] shows the extent of purification of human plasma A1PI at various stages. The procedure resulted in 48-fold purification with a yield of 12 mg protein. The amount of protein required to inhibit trypsin to the extent of 50% (I50) was found to decrease as the purification fold increased. Thus, the amount of protein required at the starting phase was 216 ?g, which was reduced to 4.5 ?g at the final phase of purification.
The molecular weight of the inhibitor as determined from HPLC was found to be 52 KD.
The extent of glycosylation of BSA, a standard protein, was estimated with TNBS and the percent of free amino groups decreased with increase in glucose concentration and also with increase in period of incubation [Table - 2]. Similar observation was made with A1PI also [Table - 3].
The molecular weight of the glycosylated inhibitor as determined by HPLC was 57 KDa.
While studying the inhibitory activity of modified (Glycosylated) A1PI, it was observed that the activity has been significantly reduced. Thus, inhibitor glycosylated with 200 mM glucose for a period of 10 days (G2D10) has I50 at 16.3 ?g protein and that glycosylated with 500 mM glucose for 10 days (G4D10) has a value of I50 at 18 ?g protein [Figure - 1]. In contrast the unglycosylated inhibitor showed the value as 4.5 ?g protein [Figure - 1]. In general, the trypsin inhibitory capacity of glycosylated inhibitor decreased with increase in glucose concentration and period of incubation [Table - 4].
Purification of A1PI from normal human plasma has been achieved using techniques of ion exchange chromatography, affinity chromatography and-gel permeation. The molecular weight of this native inhibitor was found to be 52 kD.
The plasma from diabetic patient was found to have decreased inhibitory capacity towards the proteolytic enzyme trypsin. The main inhibitors of trypsin in plasma are A2M and A1PI2. Earlier work has already shown that A2M shows decreased TIC in diabetes, the mechanism of which is not nonenzymatic glycosylation.
The mechanism of decreased TIC of A1PI was determined by the process of in vitro glycosylation of native A1PI. Uptake of glucose molecules by the native inhibitor during glycosylation could be evidenced by two facts - the increase in molecular weight from 52 kD to 57 kD and the decrease in percentage of free amino groups in the inhibitor. The term nonenzymatic glycosylation represents the interaction of glucose with specific free amino groups in proteins under mild physiological conditions not catalyzed by specific enzymes. The protein function gets altered by this process as the interaction may involve or may be close to the functional site of protein or it may change the stereochemical configuration of the protein. Some of the proteins known to have altered function due to glycation are haemoglobin10, albumin11, Ca++-ATPase12 and antithrombin III, another proteinase inhibitor of human plasma.
The most probable amino acid undergoing glycosylation is Lysine which has free amino group when present in a polypeptite chain. Importance of lysyl residues for the physiological function of A1PI was reported in studies when A1PI was treated with maleic anhydride or acetic anhydride which react with lysyl residues specifically. This treatment inactivated A1PI towards trypsin, chymotrypsin and elastase.
Thus, glycosyl modification of lysyl residues during the present work decreased its inhibitory action on trypsin.
If proteinase inhibitors like A1PI alter their normal function, limiting of unwanted proteolysis and prevention of tissue damage cannot be achieved properly. Glycosylation of proteinase inhibitors to decrease their function may partly explain some of the complications of diabetes involving eyes, kidneys and other organs that cannot be prevented by meticulous control of glycaemia.
Present study reports glycosylation of human plasma proteinase inhibitor A1PI to reduce its action of proteolytic enzyme - trypsin.
[Figure - 1][Table - 1], [Table - 2], [Table - 3], [Table - 4]