Details of Specific TLVs

Diego A. P. de Albuquerque a,c; Igor T. Cavalcanti a,c; Luydson Richardson Silva Vasconcelosc, Francisco Montenegroc; Leila Maria Moreira Beltrão Pereirad; Maria do Socorro de Mendonça Cavalcanti c; Patrícia Mourac; Eduardo Isidoro Carneiro Beltrão a,b;

Luiz Bezerra de Carvalho Júniora,b*.

a

Keizo Asami Immunopathology Laboratory (LIKA), Federal University of Pernambuco, Recife, PE, Brazil; bDepartment of Biochemistry, Biological Sciences Centre, Federal University of Pernambuco, Recife, PE, Brazil; cLaboratory of Molecular Biology of Virus, Biological Sciences Institute, University of Pernambuco, Recife, PE, Brazil; dLiver Institute of Pernambuco - IFP, Medical Sciences Faculty, University of Pernambuco, Recife, PE, Brazil.

*Corresponding author: Luiz Bezerra de Carvalho Júnior. LIKA – UFPE. Av. Prof. Moraes Rêgo, s/n, Recife, Pernambuco, Brazil. 50670-901. Telephone: + 55 81 21012504. Fax: + 55 81 21012508. E-mail: [email protected]

Abbreviations: BSA, bovine serum albumin; CRD, carbohydrate recognition domain; DAB, Diaminobenzidine; ELISA, enzyme-linked immunosorbent assay; GlcNAc, N-acetyl-D- glucosamine; HCV, hepatitis C virus; MASPs, serine-proteases associated proteins; Man, mannose; MBL, mannan-binding lectin; MM, molecular mass; PBS, phosphate buffered saline; Rc, recombinant; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; SNPs, single nucleotide polymorphisms; TBS, tris-buffered saline.

47 ABSTRACT

Mannose-binding lectin (MBL) recognizes carbohydrates via activation of the complement system and modulation of cytokine production by monocytes. Heterogeneity in molecular masses of MBL is due to the impairment of oligomers formation, which is linked to genetic polymorphisms on the exon 1 of the MBL2 gene. Individuals with genotype AA (wild type) produce high molecular mass (HMM) proteins whereas A0 and 00 ones produce intermediate (IMM) and low molecular mass (LMM) proteins. To get insight to the heterogeneous molecular mass profile of MBL, thirty Hepatitis C Virus (HCV) patients’ sera were investigated using a MBL binding assay with mannan-coated nitrocellulose (dot-N-Man). MBL obtained were evaluated by SDS-PAGE and western blotting. The molecules obtained via dot-N-Man assay showed a molecular mass profile associated to MBL after reducing and non-reducing SDS-PAGE. The molecular masses ranged from 25-270 kDa, comparable to low and high molecular mass forms of MBL. Non-reducing SDS-PAGE showed high molecular mass bands in all AA individuals while bands of 270 and 205 kDa were observed in sera of 5 out of 20 patients with A0 and 00 genotypes. The immunoblotting confirmed the MBL samples obtained from the dot-N-Man.

48 INTRODUCTION

The immune innate response is a critical determinant of organism susceptibility to infection. This is particularly important when the adaptative immunity is immature or compromised. Mannan-binding lectin (MBL) is a multimeric molecule and an important component of innate immunity which binds pathogens and activates the complement system independently of antibody. Recent studies have shown that the lectin pathway has the potential to generate four times more C3/C5 convertases than the classical pathway and hence the production of pro-inflammatory products C3a, C4a, and C5a required for mounting an effective innate immune response to infections is observed (Rawal et al., 2008). MBL recognizes mannose and N-Acetylglucosamine (GlcNAc) residues, arranged in high density on the surface of bacteria, yeast, protozoans, parasites and virus, but not the normal mammalian cells (Minchinton et al., 2002, Thiel et al., 2006). The binding leads to the change of structure and conformation of MBL, which activates serine-proteases associated proteins (MASPs) and initiates the complement cascade (Petersen et al., 2001).

The MBL2 is primarily expressed in the liver (Garred et al., 2009), the gene encodes for a 32 kDa protein chain. Three chains form a structural subunit of 96 kDa alfa-helical molecule, consisting of a homopolymer. This structural subunit is stabilized by disulfide linkages through cysteine-rich residues on the N-terminal region. MBL presents a collagen region where the MASPs bind and each chain shows a carbohydrate recognition domain (CRD) on the C-terminal region. MBL is found in different molecular forms in circulation, including single structural units, dimers, trimers and tetramers (Kilpatrick, 2003). The capacity of binding to sugars residues is highly dependent of its oligomerization state. Two or three subunits assemble to form dimmers and trimers respectively, leading to the phagocytosis of the pathogen (Cedzynski, M. et al., 2004). On the other hand MBL high molecular mass forms is ables to form complexes with MASPs and, therefore, activate the complement system, enhancing the inflammatory process (Minchinton et al., 2002; Garred et al., 2003).

MBL levels in serum as well as its functional activity have been linked to common genetic variants in the MBL2 gene. Three polymorphisms in the exon 1 affect the collagen region of MBL and give rise to allelic variants B, C and D (Lipscombe et al., 1995, Teillet et

49 al., 2005). While A means wild type allele, B, C and D are related with the structural mutations in codons 54, 57 and 52, respectively (Rema, et al., 2009). Proteins presenting these genetic polymorphisms show impaired binding to specific oligossacharides and diminished ability to MASPs association (Petersen et al., 2001). Besides the B, C and D structural polymorphisms, single nucleotide polymorphism (SNP) at promoter region has been described (Best, et al., 2009). These SNPs correspond to H/Y, X/Y and P/Q alleles in - 550, -221 and +4 gene positions. The three loci are closely related and four promoter haplotypes (LXP, LYP, LYQ e HYP) are commonly found (Madsen et al., 1998; Turner, 2003, Terai et al., 2003). In particular, codon 54 mutation of the MBL gene is associated with recurrent infections and autoimmune diseases (Tsutsumi et al., 2005; Mok et al., 2007; Gorgi

et al., 2009). In attempt to elucidate the relation between levels, function and MBL genotype

many authors have used different approaches. The most commom assay for quantify MBL is the ELISA. Mannan binding and C4 deposition assays have also been used for evaluation of functional activity of MBL and complement activation in vitro (Holmskov et al., 2003; Minchinton et al., 2002; Garred et al., 2003; Dean et al., 2005).

In the present study Hepatitis C virus sera were used and MBL molecules were screened regarding their molecular masses attempting to associate their molecular profile regarding the polymorphism in the exon 1of the MBL2 gene.

50 EXPERIMENTAL

Patient´s Samples: Thirty chronic patients positive for Hepatitis C virus (HCV) were genotyped for MBL2 polymorphism at the exon 1 region, previously to experimentation according to Segat et al., 2007, where peripheral blood DNA was extracted and MBL2 was genotyped and classified as AA, wild type (n = 10); A0, mutant type (n = 10); 00, mutant type (n = 10).

Dot-N-man: This method was modified from Rogan et al. (1991) and Romia et al. (1992). Nitrocellulose membranes (Hybond C, Amersham Pharmacia Biotech, USA) were cut with paper puncher (6 mm discs) and placed in a 96 wells microplates – one at each (Maxi Sorp, Nunc, Denmark). Mannan solution (300mMg/mL; Sigma Chemical Company, USA) in 100 mM tris-buffered saline solution, pH 7.4 with 100mM Tween 20, pH 7.4 (TTBS), was added to the membranes and allowed to dry at 25°C. After 3 washes with TTBS, membranes were incubated with albumin solution (50mg/mL; BSA, Sigma Chemical Company, USA) for 1h at 25°C. Six washes with TTBS were done and after that serum samples (1:20) in TBS-Ca++ (140 mM NaCl, 20 mM Tris, 5 mM CaCl2, pH 7.4) were incubated overnight at 4°C.

Membranes were then washed 3 times with TTBS. MBL was recovered from membranes using 1M NaCl solution or mannose solution (concentration varying from 300 mM to 600 mM). Recovered MBL molecules were pooled, lyophilized and stored at -20°C until use.

Dot-blot: lyophilized MBL was ressuspended in TBS (30 μg/μL) and 30 μL were added to 6 mm discs of nitrocellulose for immobilization by air drying. Membranes were then incubated with 100 μL of 5% BSA for 1 h at 25º. Discs were washed 6 times with TTBS and IgG anti- MBL rabbit antibody (1:300) was added and incubated overnight at 4ºC. TTBS washes (3 times) were performed and IgG anti-rabbit conjugated to peroxidase (Sigma, USA) was incubated for 1h at 25°C under gentle stirring. Diaminobenzidine (DAB)-H2O2 (10 mg/mL in

51 SDS-PAGE: Experiments were carried out according to Laemmli (1970). Lyophilized MBL was ressuspended (5μg in 20 μL) in sample buffer (Tris-HCl, pH 6.8; 20% glycerol; 4% SDS; 2% Bromophenol Blue, 5% bet a-mercaptoethanol), warmed up to 100º C in water bath for 5min and run on discontinuous gels in a SDS-PAGE system (4% stacking gel and 10% separating gel) at 75 V and 40 mA. Molecular Mass marker kit, ranging 30-200 kDa (Sigma, USA) was used. Bands were visualized with Commassie solution and silver staining.

Western blotting: SDS-PAGE bands were transferred to nitrocellulose membranes and incubated with TTBS supplemented with 5% skimmed milk solution for 90 minutes at 25°C under gentle stirring. After three TTBS washes (10 min each) primary antibody (1:100 – rabbit anti-MBL polyclonal serum – Antibody Shop, Denmark) was incubated overnight at 4º C followed by the secondary antibody (1:5000 -IgG anti-rabbit conjugated to peroxidase - Sigma, USA) for 1 h at 25°C. Diaminobenzidine (DAB)-H2O2 (10 mg/mL in PBS-T) were

used as peroxidase substrate.

Ethical Clearance: the project was approved by the Ethic Committee of the Oswaldo Cruz University Hospital at the University of Pernambuco (UPE), Northeast Brazil (no UPE/021/05 in 11/05/2005). Blood sample donors agreed in participating the project via a written consent.

52 RESULTS AND DISCUSSION

The present study used dot-N-man for MBL purification from total blood samples of HCV patients. The assay was carried out using mannan immobilized onto nitrocellulose membrane. MBL was investigated in three genotypes (AA, A0 and 00).

The separation yield of bound mannan-ligant MBL was of 4 to 8% (6% - average value) of the total blood sample (approximately 2 mg/mL). MBL specific recognition was detected and confirmed by dot-blot and western blotting.

MBL SDS-PAGE revealed differences among the three genotypes (AA, A0 e 00). AA individuals presented high molecular forms (180 kDa) as well as molecular forms of 55 to 95 kDa (Figure 1A). Low molecular form of 37 kDa related to the structural subunit of MBL, was not observed in SDS-PAGE, except for one sample of AA (Figure 1A, lane 1).

Three out of ten A0 individuals presented an electrophoresis profile similar to the wild genotype (Figure 1B, lanes 1 and 2). Bands of 55-66 kDa were observed in 00 samples but it was not observed high molecular forms or structural subunit bands (Figure 1C). Recombinant MBL was presented as one band of 55 kDa (Figure 1D, line 1) which indicates that MBL molecules separated by dot-N-man could have undergone structural alterations.

AA individuals presented high molecular forms of 97, 205, 214 and 270 kDa, where the last one is equivalent to the oligomer formed by three monomers of the lectin (Figure 2A). The structural unit of 88 kDa was absent in samples but in lane one (Figure 2A, line 1) where bands of 37 kDa and 25 kDa were also observed. In A0 and 00 individuals it was observed bands of 205, 66 and 55 kDa (Figures 2B, 2C).

Western blotting from reducing gel for AA samples revealed bands of 63 kDa, 55 kDa and 180 kDa bands.

Molecular mass analysis of human serum MBL can bring light to how this lectin functions in homeostasis and pathology. SDS-PAGE and western blotting of MBL samples showed distinct profiles for its molecular masses in the three genotypes studied. A clear difference was observed in individuals with AA genotype in comparison to the 00 homozygous variant genotype. Nevertheless, there was a considerable variation in the profiles of the heterozygous individuals (A0 genotype).

53 A key point in MBL studies is the presence of many heteroligomeric forms in human blood circulation which can form dimers to tetramers (Rajagopalan et al., 2009). This oligomerization pattern can be more complex when the allelic variants are also considered. Yokota et al. (1995) observed that, in humans and rabbits, trimers, tetramers and pentamers were the main MBL forms and that the last two exhibit complement activation action.

Population specific and non-homeostatic differences in MBL molecular mass profile were also observed by other researches (Garred et al., 2006; Lee et al., 2005). For the Korean samples Lee et al. (2005) observed that native MBL molecule presents bands raging from 50 to over 200 kDa and the molecular mass varies according to genotypes. In the present work we analyzed MBL from HVC patients in both native and reducing conditions. Under reducing conditions AA genotype MBL revealed bands of 55, 66 and 95 kDa. Under non-reducing conditions AA patients presented a broad range of bands were observed between 55 and 270 kDa in contrast to patients with A0/00 genotypes where a 205 kDa band was the main one. Such observation may be explained due to the purification process or to the great diversity of oligoforms of MBL in patients presenting wild genotype. In western blotting from native gel it was also observed a weak band of 128 kDa for one AA genotype sample, but not for A0/00 patients. Such ban is not very common to MBL being considered as anomalous monomeric form which probably presents extra peptide chains in their structure. Lipscombe et al. (1995) reported similar findings.

In the analysis of native SDS-PAGE molecular profile of 00 patients we observed a band of 205 kDa differently from what was reported by Garred et al. (2003) and Dean et al. (2005). It is known that the purification method can influence these differences. The 205 kDa band indicates the formation of a dimmer which may be responsible for the binding of MBL from A0/00 patients. Dean et al. (2005) demonstrated that depending upon the specific allele of MBL (B, C or D), the oligomerization process can be differentially affected and that the allele D exerts minor influence comparing to the others. Also the A0 genotype can generate molecules of high molecular weight due the presence of the wild allele.

Dumestre-Perard et al. (2002) questioned the possible relationship between structural polymorphism of MBL and its oligomerization state. They also reported the difficulties to evaluate MBL profile based in the protein content and genotyping. Lipscombe et al. (1995) reported that wild type MBL presented a molecular mass of 500 kDa while mutant was characterized by molecular forms of 240-380 kDa.

54 Our results indicated that MBL of both, low and high molecular masses, were identified in sera of HCV patients and that also the profile varied with the genotyping for

MBL2. The incomplete forms of MBL may be related to the diversity of oligomerization

process in either heterozygous or homozygous individuals. Also these observations are related to the molecule stability and normal or pathologic state as well as to the purification process. MBL serum concentration can be very low in normal individuals with A0/00 genotypes. In HCV patients we were able to obtain significant amounts of MBL using dot-N-man as a consequence, in part, of the chronic condition of HCV patients studied (hepatic lesion and intense inflammatory activity). Such conditions leads to higher levels of circulating MBL when compared to normal sera (Dumestre-Perard et al., 2002; Kilpatrick et al., 2003; Segat et al, 2007).

In conclusion our results depicted the molecular forms of MBL in HCV patients what could contribute to further analysis of the influence of the structure/genotype of MBL in the progression of infection and also in the response to the hepatitis C therapy.

ACKNOWLEDGEMENT

Authors thank to the Gastrohepathology Unit of Oswaldo Cruz University Hospital for blood sampling of HCV patients and for CNPq and FACEPE for financial support.

55 REFERENCES

Best, L.G., Ferrell, R.E, Decroo, S., North K.E., Maccuer, J.W., Zhang, Y., Lee, E.T., Howard, B.V., Umans, J., Palmieri, V., Garred P., 2009. Genetic and other factors determining mannose-binding lectin levels in American Indians: the Strong Heart Study. BMC Medical Genetics. 10(5), 1-8.

Cedzynski, M., Szemraj, J., Swierzko, A. S., Bak-romaniszyn, L., Banasik, M., Zeman, K., Kilpatrick, D. C., 2004. Mannan-binding lectin insufficiency in children with recurrent infections of the respiratory system. Clin. Exp. Immunol. 136(2), 304–311.

Dean, M. M., Heatley, S., Minchinton, R. M., 2005. Heteroligomeric forms of codon 54 mannose binding lectin (MBL) in circulation demonstrate reduced in vitro function. Molec. Immunol. 43(7), 950-961.

Dumestre-Perard, C., Ponard, D., Arlaud, G.J., Monnier, N., Sim, R.B., M.G. Colomb, M.G., 2002. Evaluation and clinical interest of mannan binding lectin function in human plasma. Molec. Immunol. 39, 465-473

Garred, P., Larsen, F., Madsen, H.O., Koch, C., 2003. Mannose-binding lectin deficiency- revisited. Molec. Immunol. 40, 73-84.

Garred, P., Larsen, F., Seyfarth, J., Fujita, R., Madsen, H.O., 2006. Mannose-binding lectin and its genetic variants. Genes Immunol. 7(2), 85-94.

Garred, P., Honoré, C., Ma, Y.J., Munthe-Fog, L., Hummelshøj, T., 2009. MBL2, FCN1, FCN2 and FCN3-The genes behind the initiation of the lectin pathway of complement. Mol

56 Immunol. 46(14), 2737-44.

Gorgi, Y., Sfar, I., Aouadi, H., Makhlouf, M., Abderrahim, E., Jendoubiayed, S., Bardi, R., Ben Abdallah, T., Ayed, K. , 2009. Mannose binding lectin (+54) exon 1 gene polymorphism in Tunisian kidney transplant patients. Transplant Proc. 41(2), 660-662.

Holmskov, U., Thiel, S., Jensenius, J. C., 2003. Collections and ficolins: humoral lectins of the innate immune defense. Annu. Rev. Immunol. 21, 547-578.

Kilpatrick, D.C., 2003. Introduction to mannan-binding lectin. Biochem. Soc. Trans. Vol. 31(4), 745-747.

Laemmli, U.K., Favre, M., 1973. Maturation of the head of bacteriophage T4. I. DNA packaging events. J. Mol. Biol. 80: 575–599.

Lee, S.G., Yum, J., Moon, S.H., Kim, M.H., Yang, J.Y., Kim, J.H., Yoon, L.Y., Lee, S., Song, K., 2005. Analysis of mannose-binding lectin 2 (MBL2) genotype and the serum protein levels in the Korean population. Molec. Immunol. 42, 969-977.

Lipscombe, R. J., Sumiya, M., Summerfield, J. A., Turner, M. W., 1995. Distinct physicochemical characteristics of human mannose binding protein expressed by individuals of differing genotype. Immunology 85, 660-667.

Lissilour, S., Godinot, C., 1990. Influence of SDS and methanol on protein electrotransfer to Immobilon P membranes in semidry blot systems. Biotechniques 4(397-8), 400-1.

57 Madsen, H.O., Satz, M.L., Hogh, B., Svejgaard, A., Garred, P., 1998. Different molecular events result in low protein levels of mannan-binding lectin in populations from southeast Africa and South America. J. Immunol. 161, 3169-3175.

Minchinton, R.M., Dean, M.M., Clark, T.R., Heatley, S., Mullighan, C.G., 2002. Analysis of the relationship between mannose-binding lectin (MBL) genotype, MBL levels and function in an Australian blood donor population. Scand. J. Immunol. 56 (6), 630-641.

Mok, M.Y., Ip, W.K., Lau, C.S., Wong, W.H., 2007. Mannose binding lectin and susceptibility to infection in Chinese patients with systemic lupus erythematosus. J. Rheumatol. 34, 1270-1276.

Petersen, S.V., Thiel, S., Jensenius, J.C., 2001. The mannan-binding lectin pathway of complement activation: biology and disease association. Molec. Immunol. 38, 133.

Rajagopalan, R., Salvi VP, Jensenius JC, Rawal N., 2009. New insights on the structural/functional properties of recombinant human mannan-binding lectin and its variants. Immunol Lett. 123(2), 114-24.

Rawal, N., Rajagopalan, R., Salvi, V.P., 2008. Activation of complement component C5: comparison of C5 convertases of the lectin pathway and the classical pathway of complement. Journal of Biological Chemistry. 283, 7853–63.

Rogan, M.T., Craig, P.S., Zeyhle, E., Romig, T., Lubano, G.M., Deshan, L., 1991. Evaluation of a rapid dot-ELISA as a field test for the diagnosis of cystic hydatid disease. Trans R Soc Trop Med Hyg. 85(6), 773-777.

58 Romia, S.A., Youssef, M.E., Handoussa, A.E., Rizk, H.M., Sallam, S.M., 1992. Dot-ELISA as a diagnostic test in hydatid disease.J Egypt Soc Parasitol. 22(3), 603-610.

Segat, L., Silva Vasconcelos, L. R., Montenegro de Melo, F., Santos Silva B., Arraes, L.C., Moura P., Crovella, S., 2007. Association of polymorphisms in the first exon of mannose binding lectin gene (MBL2) in Brazilian patients with HCV infection. Clin. Immunol. 124(1), 13-17.

Teillet, F., Dublet, B., Andrieu, J.P., Gaboriaud, C., Arlaud, G.J., Thielens, N.M., 2005. The two major oligomeric forms of human mannan-binding lectin: chemical characterization, carbohydratebinding properties, and interaction with MBL-associated serine proteases. J. Immunol. 174, 2870.

Terai, I., Kobayashi, K., Matsushita, M., Miyakawa, H., Mafune, N., Kikuta, H., 2003. Relationship between gene polymorphisms of mannose-binding lectin (MBL) and two molecular forms of MBL. Eur. J. Immunol. 33, 2755-2763.

Thiel, S., Frederiksen, P.D., Jensenius, J.C., 2006. Clinical manifestations of mannan-binding lectin deficiency. Molec. Immunol. 43, 86.

Tsutsumi, A., Takahashi, R., Sumida, T., 2005. Mannose binding lectin: genetics and autoimmune disease. Autoimmun Rev., 4, 364-372.

Turner, M.W., 1996. Mannose-binding lectin: the pluripotent molecule of the innate immune system. Immunol Today 17(11), 532-540.

Yokota, Y., Arai, T., Kawasaki, T., 1995. Oligomeric structures required for complement activation of serum mannan-binding proteins. J. Biochem. (Tokyo) 117, 414-419.

59

Figure 1. MBL molecular mass identified by reducirng SDS-PAGE. (A) Individuals

with AA genotype where more intense bands were visualized. Arrows show the 37 and 95 kDa bands, corresponding monomeric and dimeric forms variations. (B) Individuals with AO genotype reveals few high molecular mass forms. (C) Individuals with OO genotype. (D) A 55 kDa band of MBL recombinant was visualized in 1 (rec).

60

Figure 2. Molecular forms of MBL identified by non-reducing

SDS-PAGE. (A) AA samples with presence of a high range of molecular mass. A 25 kDa band also was observed at the number 2. (B) A0 (1,2) and (C) 00 (1,2) samples. It was observed bands with less intensity, but for all A0 and 00 samples were detected high molecular bands.

61 7 CONCLUSÕES GERAIS

- O ensaio dot-N-man mostrou-se eficiente na separação de lectina ligadora de manose (MBL) do soro de pacientes com hepatite C;

- O ensaio dot-N-man mostrou-se uma técnica simples, prática e reprodutiva para separação de formas de baixa massa molecular da MBL através do reconhecimento por anticorpos específicos, evidenciando principalmente as estruturas anômalas da lectina;

- A caracterização estrutural da MBL em relação aos genótipos (AA, AO e OO) apresentou resultados consistentes com variações pronunciadas entre os indivíduos com o gene selvagem e os que apresentaram o gene mutante, com a observação de um perfil molecular característico para cada genótipo;

- As estruturas diméricas e triméricas da MBL foram as formas predominantes nos diferentes genótipos de pacientes positivos para HCV;

- Observamos que as formas de alta massa molecular podem está associadas à fase crônica da hepatite C.