Functional estimation of mannose binding lectin associated serine protease (MBL-MASPs) in human serum.
Background & objectives: Mannose binding lectin (MBL), a C-type
or [Ca.sup.2+] dependent lectin, plays a major role in lectin pathway of
complement activation. MBL deficiency/insufficiency is associated with
susceptibility to many infections. It is important to know the
association of functional lectin levels with disease condition.
Therefore, we carried out this study to develop a simple assay to
estimate the functional MBL-associated serine proteases (MBL-MASPs)
levels in human serum samples.
Methods: A novel method was developed based on direct haemolysis of mannan coated human erythrocytes in autologous human serum for functional estimation of MBL and associated serine proteases (MBL-MASPs complex). Functional MBL-MASPs serum levels in 75 healthy individuals was estimated. Results were compared with those obtained by ELISA based assay.
Results: Lysis of mannan coated human RBC in autologous serum was highly specific and mediated by MBL-MASPs lectin complement pathway. Concentration of MBL-MASPs in serum of normal healthy individuals (n=75) was found to be 1.579 ug/ml (median= 1.149 ug/ml) by the haemolytic assay which was comparable to the values obtained by ELISA method.
Interpretation & conclusions: Our findings showed that the method developed for the estimation of functional MBL-MASPs levels in human serum is simple, cost-effective and comparable with existing ELISA method.
Key words Complement--erythrocytes--haemolytic assay--mannose binding lectin--MBL-MASPs
Lectins (Health aspects)
Monosaccharides (Health aspects)
Sugars (Health aspects)
Mannan-binding lectin (Health aspects)
Proteases (Health aspects)
Serine (Health aspects)
Enzyme-linked immunosorbent assay (Health aspects)
Gulla, Krishana Chaitanya
|Publication:||Name: Indian Journal of Medical Research Publisher: Indian Council of Medical Research Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Health Copyright: COPYRIGHT 2009 Indian Council of Medical Research ISSN: 0971-5916|
|Issue:||Date: Oct, 2009 Source Volume: 130 Source Issue: 4|
|Geographic:||Geographic Scope: India Geographic Code: 9INDI India|
Complement system provides a highly effective means for the
destruction of invading microorganisms and for immune complex
elimination (1). Complement system can be activated by three different
pathways, viz., classical pathway (CP), alternative pathway (AP), and
lectin pathway (LP). LP is activated by binding of mannose binding
lectin (MBL) or ficolin to pathogen-associated molecular patterns
(PAMPs) on the surface of microbes. Upon binding, activation of
MBL-associated serine proteases (MASP-1, MASP-2, MASP-3) takes place,
resulting in the activation of complement proteins C4 and C2 (2,3). The
C4b fragments generated, bind covalently to the microbial surface and
interact with C2 components resulting in formation of C4b2a complex. The
C4b2a complex generates opsonic C3b fragments which further activates
the complement system resulting in lysis of microbes (4,5).
MBL deficiency is quite common in human i.e., low-level haplotypes can occur in up to 30 per cent of the human population (6) and is associated with susceptibility to a variety of infectious diseases (7). Hence, it is important to estimate the functional levels of MBL for proper diagnosis and treatment.
MBL in the serum or purified samples is quantified by functional (measuring complement activation) or non functional assays (measuring protein concentration by ELISA). The anti MBL antibodies preferentially detect higher MBL oligomeric forms and thus may underestimate lower oligomers of MBL (8). The levels obtained by the ELISA have no indication of their functional activity. Functional assays are advantageous due to their potential ability to evaluate consumption of the lectin pathway components and to reveal putative defects in MBL associated serine proteases (9). Recently an MBL functional assay based on ELISA was reported (10) but use of expensive monoclonal antibody for C9 seems to be a drawback of the assay. Considering the above facts, we carried out this study to develop a simple and direct haemolytic assay to estimate the functional MBL-MASPs levels in human serum samples by calculating percentage of lysis of mannan coated human erythrocytes and compared with ELISA method.
Material & Methods
Materials: Unless otherwise stated, all reagents were obtained from Sigma-Aldrich, USA. Chromium chloride was procured from Hi-Media, Mumbai (India). Human blood was collected in Alsever's solution for isolation of erythrocytes. ELISA test kit for human MBL (Kit number: HK323) was purchased from Hycult Biotechnology (HBT), The Netherlands. Venous blood was collected from normal healthy individuals into Becton Dickinson EDTA coated vacutainers and the plasma recalcified to make normal human serum (NHS). Samples collected from healthy individuals males 50; females 25; age 20-55 yr (between 2007 to 2008 at Indore, Madhya Pradesh, India) to measure MBL concentration for another study (MBL exon1 polymorphism) were used for this study also. Sample collection was approved by the Ethical Committee, School of Life Sciences, Devi Ahilya Vishwavidhyalaya, Indore. Written consent was taken from the volunteers.
Preparation of mannan coated erythrocytes: Human blood collected in Alsever's solution subjected to centifugation at 600xg to collect erythrocyte. Erythrocytes were washed with gelatin (0.1%) containing 5 mM HEPES buffered saline (pH7.4) (GHB) and diluted to 1x[10.sup.9] cells/ml. One ml of erythrocytes (1X[10.sup.9] cells/ml) was incubated with 1ml of chromium chloride (3 mg/ml) and 1 ml of mannan (2 mg/ml) solution for 5 min at 25[degrees]C (11,12). After incubation, the cells were washed three times with ice cold Gelatin HEPES buffer saline containing 2 mM Ca[Cl.sub.2] and 0.5 mM Mg[Cl.sub.2] ([GHB.SUP.2+]) and used for the haemolytic assays. Before the assay, the blood group of each sample was also determined as serum was subjected to haemolytic assay using the mannan coated RBCs from the respective blood group only.
MBL-MASPs mediated complement assay: Initially for optimizing the parameters mannan coated erythrocytes (100[micro]l of 1X[10.sup.8] cells/ml) were incubated with serially diluted human serum (100 [micro]l) at 37[degrees]C for 60 min. After standardization, all the experiments were done with 1:2 dilution of human serum. Controls included both normal erythrocytes and chromium chloride treated erythrocytes incubated with serum. After incubation, the cells were centrifuged at 600 x g and haemoglobin released in supernatant was measured by using ELISA plate reader (Multiskan MS, India) at 414 nm. Pooled normal human serum was passed through the Mannan-Sepharose column at 4[degrees]C. The flow through was used as MBL deficient serum (13). Addition of mannan-coated erythrocytes to this serum did not result in lysis indicating that all MBL has been removed. Purified MBL-MASPs was isolated by affinity chromatography on mannan agarose essentially as described by Matsushita et al (14). Purified MBL-MASPs was then estimated by HBT MBL ELISA kit. Purified MBL-MASPs was added to deficient serum to check for the recovery of haemolytic activity. Calibration curve was made between the percentage of recovered haemolytic activity by deficient serum and amount of purified MBL added. Amount of MBL-MASPs in test serum was calculated using the equation derived from above calibration curve. To remove anti-mannan antibodies serum was passed through the Protein-G Hitrap column (Pharmacia) at a flow rate of 0.2 ml/min. Removal of antibodies was confirmed by the absence of a major band on SDS PAGE. Flow through serum was subjected to MBL-MASPs mediated haemolytic complement assay.
Effect of sugars, chelators, and inhibitors on MBL-MASPs mediated complement assay: Purified MBL-MASPs were incubated with mannan coated RBCs in presence of the sugars and chelators such as mannose (1.5%), glucose (1.5%), Ca[Cl.sub.2] (2 mM), EDTA (10 mM), Mg[Cl.sub.2]-ethylene glycol tetraacetic acid (EGTA) (0.5 and 10 mM respectively) and lytic activity was checked. In the case of enzyme inhibitors, MBL-MASPs bound mannan coated RBCs were incubated with Pefabloc (1 mM), phenylmethanesulph onylfluoride (PMSF) (2 mM), boroMpg (0.1 mM), Cl inhibitor(1 and 10 [micro]M) at 4[degrees]C for 20 min with intermittent shaking. After washing, the cells were taken and MBL deficient serum was added as source of complement components to check the remaining lectin activity.
Estimation of MBL concentration in the serum of normal healthy individuals by using ELISA kit was done as per manufacturer's instruction. Standard curve was made between 0.41 to 100 ng/ml of MBL. Blood group determination was done by using Anti A, B, D monoclonal antisera from Span Diagnostic Ltd, India, as per manufacturer's instructions.
Inter-assay and intra-assay coefficients of variation were estimated from 10 measurements of NHS using the equation (s / x) X 100 per cent, where s is the sample standard deviation and x the sample mean.
Statistical analysis: Spearman correlation test was used for the correlation between the data obtained by MBL ELISA and lysis assay developed. Data were analysed using "Analyse-it" statistical software (Analyse-it Software Ltd., UK).
Mannan coated erythrocytes were 98 per cent lysed when they were exposed to autologous serum whereas the normal and chromium chloride treated erythrocytes exhibited no lysis in autologous serum (Fig. 1). Lysis was completely absent in case of deficient serum prepared by passing the NHS through the mannose-Sepharose column. There was no difference in percentage of lysis when the serum was passed through the Pharmacia Protein G Hitrap Column (data not shown) showing that the antibodies did not contribute in the lytic activity in the present assay.
Presence of Mg[Cl.sub.2] and Ca[Cl.sub.2] in working buffer led to increase in lysis by only 5 per cent whereas lysis was completely inhibited by Mg-EGTA and EDTA proving that the lysis was via MBL mediated pathway only as MBL binds to mannan on pathogen surface in a calcium dependent manner (7). Lysis was completely inhibited by mannose but not by glucose. Lysis was also completely inhibited when serine protease inhibitors Pefabloc, PMSF and C1 inhibitor (C1-INH) were preincubated with RBC bound MBL-MASPs (Fig. 2).
The lytic activity was recovered up to 70 per cent when deficient serum was supplemented with purified MBL-MASPs. This in turn proved that the lysis was based on MBL lectin complement pathway only. A calibration curve was prepared between the percentage of lytic activity recovered by the MBL deficient serum against the concentration of externally added MBL-MASPs (Fig. 3). The equation derived from the calibration curve was used to calculate the MBL concentration in test serum obtained from normal healthy individuals and the results were compared with the data obtained by quantitative ELISA for MBL (Fig. 4).
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
Mean MBL serum concentration in normal healthy individuals estimated by ELISA was found to be 1244.77 ng/ml [+ or -] 977.57 (mean [+ or -] SD) whereas that of MBL-MASPs by the RBC lysis method was found to be 1579.51 [+ or -] 1180.63 ng/ml (mean [+ or -] SD). The difference was not statistically significant (Table).
Regression analysis was done to find the correlation between the data obtained by both methods and the R2 was found to be 0.9307, which showed the reliability of the present method with the established ELISA method. Intra-assay variation was 6.18 per cent and the inter-assay variation was 5.81 per cent (Fig. 5).
Lysis of mannan coated RBCs occured when exposed to autologous serum but chromium chloride treated control cells or untreated RBC did not exhibit lysis. The lysis was inhibited by mannose, EDTA and EGTA. These results confirm that the lysis occurs via MBL-MASPs mediated lectin complement pathway. Incomplete inhibition of haemolytic activity by boroMpg, a MASP-1 inhibitor showed that lysis of mannan coated RBC via complement pathway was mediated by MASP-2. This is in agreement with the fact that it is MASP-2 and not MASP-1 that activates lectin complement pathway by cleaving C2 and C4 (2).
[FIGURE 4 OMITTED]
[FIGURE 5 OMITTED]
We found decreased lectin pathway activity whenever plasma was used instead of serum from the same individual and our observations were in accordance with Seleen et al (10). The probable cause of decreased activity may be the dissociation of MBL-MASP complexes in a calcium free environment, which re-associate very slowly (2).
Haemolytic assays have already been developed for the estimation of MBL levels in human serum by several researchers but these have their own disadvantages. A haemolytic assay was developed based on the principle of yeast induced lysis of bystander chicken erythrocytes for the estimation of functional mannose binding lectin levels in human serum (15). This assay was proved to be tedious because of multi-steps and complex reagents. Another assay was developed by Suankratay et al (11), for estimation of lectin pathway of complement activation based on lysis of MBL sensitized mannan-coated sheep erythrocytes by human serum. Requirement of purified MBL and possible opsonization by antibodies against sheep erythrocyte membrane antigen present in human serum are disadvantages of this assay. The present method overcomes the above problems. Blood grouping is not an additional burden in the case of the developed assay as it could be easily done.
One of the authors (KG) acknowledge the University Grants Commission for Junior Research Fellowship (no. F-2-56/2002/ (I) EU-II). Financial assistance from DRDO/LSRB -39/EPB/2002 grant is gratefully acknowledged.
Received May 2, 2008
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Reprint requests: Dr Krishana C. Gulla, Associate Scientific Manager, Research & Development, BIOCON Limited 20th K. M. Hosur Road, Electronic City, Bangalore 560 100, India e-mail: email@example.com, Krishana.Gulla@biocon.com
Krishana Chaitanya Gulla *, Kshitij Gupta * & Krishnan Hajela *, **
* School of Life Sciences, Devi Ahilya University, Indore, India & ** School of Biological Sciences University of Southampton, Southampton, United Kingdom
Table. MBL serum concentration (ng/ml) estimated by ELISA and MBL-MASPs concentration (ng/ml) estimated by RBC lysis assay N Mean SD SE 95% CI of mean ELISA 75 1244.77 977.57 112.880 1019.85 to 1469.70 RBC lysis 75 1579.51 1180.63 136.327 1307.88 to 1851.15 Median IQR 95% CI of median ELISA 866.677 713.82 775.75 to 1049.70 RBC lysis 1149.18 947.72 947.72 to 1386.86
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