Mannose-Binding Lectin in the Innate Immune System [1 ed.] 9781614702078, 9781606927168

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Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved. Mannose-Binding Lectin in the Innate Immune System, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved. Mannose-Binding Lectin in the Innate Immune System, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook

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MANNOSE-BINDING LECTIN IN THE INNATE IMMUNE SYSTEM

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Mannose-Binding Lectin in the Innate Immune System, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved. Mannose-Binding Lectin in the Innate Immune System, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook

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MANNOSE-BINDING LECTIN IN THE INNATE IMMUNE SYSTEM

IARA DE MESSIAS-REASON AND

ANGELICA B. W. BOLDT

Nova Biomedical Books New York

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All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com

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NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA ISBN: 978-1-61470-207-8 (eBook) Available upon request

Published by Nova Science Publishers, Inc.  New York

Mannose-Binding Lectin in the Innate Immune System, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook

CONTENTS

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Preface

vii

Chapter 1

Introduction

1

Chapter 2

MBL2 Association with Diseases

25

Chapter 3

Conclusion

43

References

45

Index

77

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PREFACE Mannose-binding lectin (MBL) is a plasma protein with an important role in the innate immune system. MBL recognizes pathogens through carbohydrate structures present on the surface of a range of pathogenic organisms including viruses, bacteria, fungi and protozoans. These structures may be referred to as pathogen-associated molecular patterns (PAMPs). After binding to PAMPs, MBL promotes C1- and antibody-independent activation of complement, leading to complement-mediated killing and/or phagocytosis. MBL is also known to modulate the secretion of cytokines from macrophages and to mediate the clearance of apoptotic cells as such playing a role in the inflammatory response. The concentration of MBL in plasma varies significantly among different individuals and is determined genetically by single nucleotide polymorphisms (SNP) in the first exon and in the promoter region of the MBL gene (MBL2). Three SNPs in codons 52 (Arg52Cys, allele D), 54 (Gly54Asp, allele B) and 57 (Gly57Glu, allele C) of exon 1 significantly alter the serum concentration and biological function of MBL, leading to a deficiency in homozygous individuals. In addition, polymorphisms in the promoter region of the MBL2 gene (H/L, X/Y, P/Q, at positions -221, -550 and +4, respectively) are also known to affect MBL concentration. MBL deficiency represents a recently recognized hereditary immunodeficiency of innate immunity, affecting between 10 to 20% of individuals. The biological significance of MBL is primarily indicated by the clinical consequences of its deficiency. Low producing MBL2 genotypes and low MBL concentration have been found to be associated with predisposition to a range of infections, notably by extra cellular pathogens, and to some autoimmune diseases. MBL deficiency has also been shown to influence the progression and disease severity of different disorders, including HIV infection, rheumatoid

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arthritis and cystic fibrosis. On the other hand, MBL deficiency has been reported to be beneficial in infections due to intracellular pathogens, such as Mycobacterium leprae and Leishmania chagasi, which use C3 opsonisation and C3 receptors to invade host cells. Recently, increasing evidence has indicated a proinflammatory role for MBL in chronic diseases and situations where there is excess complement activation and tissue injury. This chapter will summarize the atual understanding of human MBL biology and introduce the general aspects of the structure, function and genetics of MBL, as well as an analysis of the role of MBL in the predisposition to clinically relevant diseases.

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Databases: MBL2 – OMIM: 154545; GenBank: AF080508, Y16576, Y16577, Y16578, Y16579, Y16580, Y16581, Y16582; NCBI Protein: NP_000233; Gene Cards: GC10M054195; dbSNP: 4153.

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Chapter 1

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INTRODUCTION The innate immune response, as the first response of the host to invading pathogens, provides important effector mechanisms operating in conjunction to eliminate invading microorganisms. This response is also known to have significant influence in determining the nature of the subsequent protective adaptive immune response. When pathogens break the physicochemical host barriers they are immediately intercepted by an array of humoral and cellassociated innate proteins targeting them for destruction and removal. One of these humoral proteins is the mannose-binding lectin (MBL), also referred to as mannan-binding lectin or mannan-binding protein [1].

1. MANNOSE BINDING LECTIN (MBL) CHARACTERISTICS MBL belongs to a family of proteins called collectins, which have a similar modular domain architecture consisting of four regions; a cysteine-rich Nterminal domain, a collagen-like region, an alpha-helical coiled-coil neck domain and a C-terminal carbohydrate recognition domain (CRD). The term “collectin” originates from the names of the two major domains shared in the polypeptide: collagen and lectin [1]. MBL recognizes sugar moieties via a Ca2+-dependent mechanism and is therefore classified as a C-type lectin. It is sometimes also referred as defense collagen, due to the immune functions carried out by the collagen domain [2].

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The other major human collectins, surfactant protein A1 and 2 (SP-A1 and 2) and surfactant protein D (SP-D), possess structural characteristics similar to those of MBL and are found predominantly in the lung and other mucosal sites [1]. A structurally and functionally related protein family known as the ficolins, also has lectin domains attached to collagenous regions (reviewed by [3,4,5], however, their CRD is a fibrinogen-like domain. Three human proteins belong to this family: L-ficolin, M-ficolin, and H-ficolin. L- and H-ficolins are humoral factors synthesized by hepatocytes. In contrast, M-ficolin is found on peripheral blood mononuclear cells, polymorphonuclear cells and type II lung epithelial cells. They are considered to have different binding specificities when compared with MBL [1].

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1a. MBL2 Gene and Protein Structure There are two MBL genes in mammals, which are most likely due to a gene duplication event [6]. Nevertheless while rodent MBL is the product of two separate genes encoding serum MBL-A (mbl1) and liver MBL-C (mbl2), humans and higher primates have an expressed pseudogene (MBL1P1) and a functional gene (MBL2) [7,8]. In fact, MBL2 is known to encode the human serum and liver MBL forms [9,10]. The MBL2 gene is located on chromosome 10q11.2-q21 [11]. The other human collectin genes encoding SP-A1, SP-A2 and SP-D - SFPTA1, SFTPA2 and SFTPD - occur sequentially just downstream from MBL2, as the MBL1P1 pseudogene (10q22Æq23) [12,13]. MBL2 contains five exons (Figure 1). Exon 0 is not translated. Exon 1 encodes the signal peptide, the N-terminal region of the protein and the first part of the collagen domain. The signal peptide has the typical hydrophobic structure of a secreted protein. The N-terminal sequence is a stretch of 21 residues of indeterminate structure, but with three important cysteine (Cys) residues involved in interchain dissulfidic bonds. The first part of the collagen domain displays seven typical Gly-Xaa-Yaa repeats (where Xaa and Yaa are any amino acid apart from glycine, Yaa is mostly proline or lysine). The small glycine residue at every third position actually makes the tightly twisted collagen-like structure possible. The collagen coding sequence is interrupted just before the first intron by the sequence Gly-Gln-Gly, giving rise to a kink in the collagen structure [14]. Exon 2 encodes the rest of the collagenous domain, with 12 additional Gly-Xaa-Yaa repeats. The collagenous region is well conserved among non-human primates [15] (Figure 2).

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Figure 1. Relationship of MBL2 exons with MBL domains (based on [31,11]. Exons are represented by boxes, untranslated regions by fine lines, translated regions by thick lines. The size of exons and introns is given in base pairs (bp). GRE: glucocorticoid responsive element. HNF3: hepatocyte-specific nuclear factor 3. HSP: homologous sequence to the promoter of heat shock protein. CRD: carbohydrate recognition domain.

The neck region is encoded by exon 3 and forms an α-helical coiled-coil that serves as polymerization site of the polypeptides [16]. The CRD and an untranslated region (UT) of variable size (310 – 2500bp) is encoded by exon 4 [11,17,14]. The UT region can contain seven ATTTA repetitions, which can destabilize the mRNA and lead to rapid degradation [11]. The CRD has a conserved globular structure of 148 amino acids, with two Ca2+ ligand sites [18]. Sugar binding involves hydrogen bonds to four amino acid residues and coordination bonds to Ca2+ (Figure 2). MBL does not just selectively bind mannose or its multimers, but rather recognizes sugars with 3- and 4-OH groups placed in the equatorial plane of the sugar ring structure [19] [20], such as glucose, L-fucose, N-acetyl-mannosamine (ManNAc) and N-acetyl-glucosamine (GlcNAc), and not galactose. Oligomerization occurs through disulphide bonds between three identical 25kDa primary subunits that become 32 kDa after glycosylation. These three protein subunits associate to form a 96 kDa triple helical MBL structural unit [21] (Figure 3). Trimerized, the CRD of rat mbl has a maximal diameter of 8 nm, and the collagen tail with the other domains sum approximately 25 nm [22]. Further disulphide bonds are formed between each structural unit to form MBL oligomers [23]. The oligomeric forms of MBL have different functional activities and circulate in human plasma as single structural units called monomers, to higher

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order oligomers up to octamers [24,25]. Large oligomeric forms of MBL (tetramers and higher) are the major type present in circulation [26,24,27,25]. The overall structure of MBL hexamers resembles that of C1q [1]: bouquet-like structures with rod-like collagen stems topped by globular CRDs [28] (Figure 3).

Figure 2. Amino acid sequence of human MBL (modified from [10]. : excision point of the signal peptide (E is the N-terminal residue of mature MBL). ‘: probably hydroxilated [327]. Within brackets: allelic MBL2 variants (MBL2*F in codon 12, E in codon 25, S39N in codon 39, S40N in codon 40, D or R52H in codon 52, B in codon 54 and C in codon 57; N176S and E209stop in exon 4 [126,106,136,133,134]. In italics: Gly residues of the GlyXaa-Yaa triplets [11]. ↓: interruption of the Gly-Xaa-Yaa triplet sequence, typical of nonfibrillar collagen [11]. Different underline styles: beginning and end of exon sequences (dashed exon 1; double exon 2; dotted exon 3; simple exon 4) [17]. In bold: conserved residues among primates [15]. ∇: Matrix metalloproteinase (MMP) cleavage sites of variant MBL: 2 (MMP-2), 9 (MMP-9), and 14 (MMP-14) [143]. Within parentheses: Ca+2 ligand residues (E212 and N214, E220 and N232 also form hydrogen bonds with the 3-OH and 4-OH groups, respectively, of mannose and other sugar residues) [20]. Squared: critical sequence in mediating phagocytosis through C1qR1p [97].

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Introduction

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Figure 3. MBL maximal polymerization. Black circles represent Ca+2 binding sites in the globular CRD. Dashed: coiled coil neck region. Thick line: collagenous region. Curved: Cys N-terminal region.

The oligomeric configuration of the structural units allows the MBL molecule to have multiple CRDs, facilitating multivalent, high-avidity ligand binding [1]. The affinity of a CRD for one monosaccharide is weak, in the order of 10-3 M. Yet the MBL avidity for highly glycosylated albumin was estimated at about 10-9 M [29]. The discrimination of self from nonself by MBL is dependent on two main factors: (1) most carbohydrate structures in animals are terminated by unrecognized sugars, e.g., galactose or sialic acid, and (2) mammalian cells normally do not present the pathogen-associated molecular patterns (PAMPs) characteristic of microorganisms, since most of the sugar residues of mammalian glycoproteins and glycolipids are separated by 20 – 30 Ǻ and cannot cross the 45 – 53 Ǻ distance between the oligomerized CRDs [30].

1b. MBL2 Transcription and Translation The transcription of MBL2 is regulated by two alternative promoters (named 0 and 1) where promoter 0 derived transcripts include an additional 5’ untranslated region encoded by an extra exon (exon 0). Alternative transcription therefore results in different lengths of mRNA transcripts that are encoding an identical MBL protein. Both promoter sequences includes TATA box for transcription initiation and DNA sequences recognized by different transcription factors: interleukin (IL)-6 (typical of acute phase protein promoters), hepatocyte-

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specific nuclear factor (HFN)-3 (involved in liver specific transcription) [31], glucocorticoids (GRE) and heat shock proteins (HSP) (Figure 4) [17].

Figure 4. MBL2 promoter 1 and exon 1 sequence with published variants. The sequence corresponds to the allelic haplotype MBL2*LXPA (Genbank.Y16580) [17]. Official numbering follows the Y16580 sequence, negative/positive numbers within parentheses decrease/increase from the second alternative transcription site and are commonly used to designate nucleotides from the promoter and exon 1 region, respectively [132]. Arrows pointing forward: alternative transcription sites. [31,11,17]. Arrows pointing upward: alternative acceptor splice sites [44]. High case, underlined: regulatory sequences (from up to down: two glucocorticoid responsive elements (GRE), one heat shock protein consensus promoter sequence shared with IL-6 responsive element type 1, another GRE and one consensus sequence for HNF3, CAAT box and two TATA boxes) [31,11]. Bold, underlined: SNP and deletion (PNG SNPs found in Papua New Guinea [131], GB synonimous variant [130], AF rare non-synonimous SNPs found in Africa [106,133,134], rs… and ss… found only in the SNP database, letters within parentheses are common names for the most studied variants [136,132]. Low case: introns and upstream regulatory region (promoter 1). High case: exon 1 translated sequence.

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Introduction

7

The first evidence that MBL is an acute-phase reactant was given by Ezekowitz et al. in 1988 [14]. The authors found that MBL mRNA transcripts were barely detectable in normal liver but that induction was seen in liver exposed to acute stress. Subsequent studies have shown that MBL levels can increase between 1.5 and threefold during the acute phase [32]. Nevertheless genetic polymorphisms can cause interindividual variations in MBL levels up to ten fold, even during the acute phase [33]. Arai et al. demonstrated that MBL2 expression is up-regulated by acute phase mediators, such as interleukin (IL)-6, dexametasone and heat shock [34]. Other in vitro studies demonstrated that cells exposed to medium with growth hormone (GH), IL-6 or thyroid hormones (T3 or T4) presented significantly increased MBL synthesis in a dose-dependent manner, however IL-6 caused a weak increase in MBL production compared to the hormones [35]. The effect of GH on MBL levels was further confirmed in two different studies of hormone replacement therapy [36,37]. MBL is mostly expressed in the liver, but it has also been detected in extravascular milieux such as synovial fluid [38], amniotic fluid [39], lung [40] and vaginal [41,42] lavages and human milk [43]. Low extrahepatic transcription on mucosal surfaces has furthermore been reported in small intestine and testis tissue, where the mRNA level comprised about 1% and 0.2%, respectively, of that seen in liver [44]. The majority of transcripts are thus derived from MBL2 promoter 1 and varied due to the use of alternative acceptor splice sites located inside exon 1 (Figure 4) [31,44]. During pregnancy, MBL concentrations increase to 140%. This increase is already present at 12 weeks of pregnancy and directly post-partum MBL concentrations drop to 57% of baseline [45]. Increasing serum MBL levels during early childhood after birth have been observed in different populations [46,47,48,49]. Serum MBL appears to reach its highest levels in a human lifetime within 5 days after birth [48] or at the age of 1 month [46] and appears to decline in levels until early adulthood [50,51]. In human milk, MBL concentration decreases significantly during development from colostrum (550 +/- 90 ng/ml) to mature milk (170 +/- 20 ng/mL). The high levels observed during the first days of lactation support the hypothesis that MBL plays a key role in limiting the colonization of the newborn gut by pathogens [43], and the high concentrations of MBL in infants suggests a critical role of this molecule in the vulnerable period of infancy before adequate specific immune protection is established [52]. Polypeptide chain trimerization occurs just after translation in the rough endoplasmic reticulum [22], beginning with the Cys residues of the N-terminal domain and generating a collagen triple helix [53,54]. In the Golgi complex, the Pro and Lys residues of the collagen part become hydroxilated and various

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Iara de Messias-Reason and Angelica B. W. Boldt

hydroxilysines become glycosilated [55] (Figure 2). Hydroxiproline stabilizes the triple-helix, and hydroxilysine, the crossed inter- and intra-chain collagen bonds [56]. Different MBL oligomers probably arise because of the way in which they assemble within the endoplasmic reticulum of liver cells during biosynthesis. Pulse-chase experiments show that while individual rat MBL subunits assemble rapidly, the protein matures into the larger oligomeric forms more slowly as it moves to the cell surface for secretion [57]. A key factor controlling the sizes of oligomers appears to be the pattern of disulphide bonds at the N-terminal ends of subunits. Each polypeptide in human MBL has three cysteine residues within this region that can potentially form disulphide bonds. Certain bonding patterns allow interchain bonds to two additional subunits and are therefore compatible with further oligomerisation, whereas other patterns permit linkage to only one subunit, therefore serving as ‘terminating patterns’ [58].

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2. MBL FUNCTIONS MBL acts as a pattern recognition receptor (PRR), recognizing PAMPs or ACAMPs (apoptotic cell-associated molecular patterns) represented by arrays of repetitive carbohydrate structures on the surface of a range of pathogenic organisms including viruses, bacteria, fungi, protozoans and multicellular parasites (Table 1), and on apoptotic cells. It can further recognize phospholipids [59], nucleic acids [60] and non-glycosylated proteins [61]. Neth et al. used flow cytometry to demonstrate MBL binding to clinically relevant bacterial isolates from immunocompromised children and noted differences in binding within some species [62]. The role of specific structural features of microorganisms (e.g. the capsule), which permit or prevent binding to MBL, has been explored in several studies. The earliest significant work was by Kawakami et al. on the so called RaRf complex (later identified as MBL) and its interaction with Salmonella enterica serovar Typhimurium [63], suggesting that the structure and composition of lipopolysaccharide (LPS) play a crucial role in MBL binding and function. Lipooligosaccharide sialyation also enable microorganisms to avoid recognition and killing by MBL [64,65]. Despite much progress in this area, certain aspects deserve further investigation, as for example the exact disposition of sugars on microbial surfaces [66]. After binding to targets, MBL induces several biological effects such as complement activation, complement-independent opsonophagocytosis, modulation of inflammation, recognition of altered self-structures and apoptotic

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Introduction

9

cell clearance (Figure 5). Since it is a pleiotropic molecule, MBL deficiency can have an impact on many different areas of the innate immune response. Some of its most important functions will now be explained in detail.

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Table 1. Selected microorganisms that have been shown to bind MBL (adapted from [66]) Bacteria Actinomyces israelii Bifidobacterium bifidum Burkholderia cepacia Chlamydia pneumoniae Escherichia coli Haemophilus influenzae Klebsiella aerogenes Leptotrichia buccalis Listeria monocytogenes Mycobacterium avium Mycoplasma pneumoniae Neisseria meningitidis Pneumocystis carinii Proprionibacterium acnes Pseudomonas aeruginosa Salmonella montevideo Staphylococcus aureus Streptococcus pneumoniae Viruses Influenza A HIV Herpes simplex 2 SARS-CoV Fungi Aspergillus fumigatus Candida albicans Cryptococcus neoformans Protozoa Cryptosporidium parvum Leishmania (Viania) braziliensis Plasmodium falciparum Trypanosoma cruzi Multicellular parasites Shistosoma mansoni

Reference [311] [311] [312] [223] [313] [62,313] [62] [311] [313] [314] [179] [62,313] [315] [311] [312] [96] [62] [62] [316,317,62] [318,319,320] [321,322] [182] [62] [62,184] [185] [323] [253] [324] [325] [324]

HIV, human immunodeficiency virus; MBL, mannose-binding lectin; SARS-CoV, severe acute respiratory syndrome–coronavirus. Mannose-Binding Lectin in the Innate Immune System, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook

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Figure 5. Some MBL functions. The lectin pathway of complement is activated by MBL and ficolins. On binding to appropriate targets, MBL–MASPs complexes cleave C4 and C2 to form C3 convertase (C4bC2b). MBL–MASP1 complex may activate C3 directly. This results in the microorganisms becoming covered with activated fragments (C3b, iC3b, C3d) of complement factor C3, leading to increased phagocytosis and creation of the membrane attack complex (MAC) which may cause lysis of the microorganism. MBL, mannose-binding lectin; MASP, MBL-associated serine proteases.

2a. Activation of Complement The complement cascade is a fundamental component of the immune system, providing protection against invading microorganisms through both antibodydependent and -independent mechanisms [67]. It also mediates many cellular and humoral interactions within the host, including chemotaxis, phagocytosis, cell adhesion and B-cell differentiation, thereby helping to coordinate and direct an effective immune response [68]. Three different pathways initiate the complement cascade: the classical, alternative and lectin pathways. A role for MBL in host defence was first proposed in 1987 when Ikeda et al. observed that the protein was able to activate the classical pathway of complement [69]. However, it is now clear that MBL activates a third pathway of complement, called the lectin pathway, in an antibody- and C1-independent fashion (Figure 6). Ficolins can also activate the same pathway in complexes with MBL associated serine proteases (MASPs) [4]. In humans there are three of these, MASP-1, -2, and -3, along with an alternative splicing fragment of MASP-2 called MAp19 or

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Introduction

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sMAP [70]. The three enzymes have the same modular buildup as C1r and C1s of the classical pathway, i.e., they are composed of well-described domains in the order: CUB1-EGF-CUB2-CCP1-CCP2-SP. The first five domains constitute the A-chain, and the serine protease SP domain constitutes the B-chain. MASP-1 and -3 are alternative splicing products of the MASP1 gene [71,72]. They share the same A-chain, apart from the 15 C-terminal residues, which constitute the linker or activation peptide, but have distinct B-chains. MAp19 and MASP-2 are also alternative splicing fragments of one gene, the MASP2 gene, with MAp19 being a smaller product consisting of only CUB1-EGF with an additional four unique amino acids at the C-terminal [73,74]. The three MASPs and MAp19 are all able to homodimerize and associate with MBL, H- and L-ficolin, in the presence of Ca2+, but apparently do not form heterodimers [75,76]. MBL dimers, trimers and tetramers of the structural subunit can bind with high affinity to at least one MASP dimer. Although single MBL subunits also bind to MASPs, the affinities are 1000-fold lower than the larger MBL oligomers [75]. While MASP-2 is able to cleave C4 with high efficiency, and C2 to a lower degree, on its own activating the lectin pathway [77], MASP-1 has also been reported to be able to cleave C2 [75,78,79]. MASP-1 and -2 thus probably cooperate in the activation of the lectin pathway [80]. Upon the binding of MBL to its PAMP targets a conformational change probably occurs, causing autoactivation of associated MASP-1 and -2 [81]. Autolysis occurs at a single site within the short linker region between the CCP2 module and serine protease domain. The active protease domain remains attached to the N-terminal fragment through a single disulphide bond [82]. MASP-2 then mainly works on C4 to generate C4b, while MASP-1 provides an increased amount of C2b for the C3 convertase [83]. Some studies also find that MASP-1 has direct C3 activating capacity [84,78], although the biological significance of this activity is not certain. The classical, alternative and lectin activation pathways converge in the common lytic cascade, which terminates in the formation of the membrane attack complex (MAC), also known as the terminal complement complex (TCC). The MAC inserts into cell membranes and forms pores leading to osmotic lysis of the target cells (Figures 5 and 6). The alternative pathway serves further as an amplification loop for the classical and lectin activation pathways [85,86]. In addition to the standard pathways, several so-called weaker bypass pathways of complement activation exist, which can come into play when some components are missing, although at a slower rate. There is a MBL-dependent C4bypass pathway [80,87,88], where high MBL concentrations, after PAMP recognition, can support C3 deposition in the absence of C2, C4 and MASPs. A more efficient lectin C2-bypass pathway, which would then be dependent on C4

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and the C4-cleaving MASP-2, also seems to occur [86]. MBL further alters the nature of the C3 acceptor bond, resulting in an amide linkage between C3 and putative bacterial acceptor molecules, possibly by altering the bacterial surface and allowing access of C3 to bacterial outer membrane proteins [87]. The change from hydroxyl acceptor to amine acceptor could be directly caused by the binding of MBL to the sugar compounds containing hydroxyl groups, promoting the reaction of C3 with the available amines [80].

Figure 6. The lectin pathway of the complement system (based on [80]), which can be activated by MBL or ficolins. Main activators are shown in square boxes, proteins of the pathways in rounded boxes, and released fragments as plain text. In italics, active serine proteases. The dashed arrows progressing from MASP-1 indicate activities on which there is not yet consensus. PAMP pathogen-associated molecular patterns; MBL mannose binding lectin; MASP MBL-associated serine protease; MAC membrane attack complex.

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Introduction

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as fluid-phase regulation. Specific control of the classical and lectin pathways is achieved through C1-inhibitor (C1-inh), a member of the serpin family of inhibitors, and human neutrophil peptide-1 (HNP-1), a member of the α-defensin family [89,78]. C1-inh forms a covalent 1:1 complex with both the activated MASP-1 and -2, limiting activation of complement [78]. The lectin pathway is more potently inhibited than the classical pathway at low doses of C1inh [90].

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2b. Opsonophagocytosis Sixty years ago, Frank Burnet and John McCrea identified three inhibitors able to inactivate influenza virus (reviewed by [66]). The β-inhibitor was recognized only in 1990 to be MBL [91]. Opsonization defects due to MBL deficiency were also recognized long before the discovery of the underlying problem. In 1968, Miller et al. reported a plasma-associated defect of phagocytosis in a child with severe recurrent infections [92]. In vitro work revealed a failure of the child’s plasma to opsonize Saccharomyces cerevisiae. This defect was later detected in the sera of children with recurrent unexplained infections [93] and chronic diarrhoea of infancy [94], but studies in the general population also revealed a relatively high frequency of the defect (5%). In 1981, this opsonic deficiency was linked to the complement system by demonstrations that sera with the deficiency deposited less C3b on yeast surfaces [95]. However, it was not until 1989 that the common opsonic defect was found to be associated with low levels of the mannose-binding protein, known now as MBL [52]. Soon after, Kuhlman et al. reported that MBL was able to interact directly with cell surface receptors and promote opsonophagocytosis of Salmonella montevideo [96]. Presently it s known that MBL and C1q enhance FcR-mediated phagocytosis through a conserved GE(K/Q/R)GEP motif in the collagen region by both monocytes and macrophages in vitro and stimulate CR1-mediated phagocytosis [97,98] (Figures 2 and 5). This process is mediated by the surface glycoprotein C1qRp (or CD93), which normally occurs on phagocytes and endothelial cells [99,98]. The mechanism by which CD93 influences these phagocytic activities remains to be defined. Other possible MBL receptors on phagoytic cells include cC1qR/calreticulin (complexed with CD91) [100], the CR1 (or CD35) receptor [101] and the integrin α2β1 on peritoneal mast cells [102].

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2c. Modulation of Inflamation An adequate modulation of proinflammatory cytokine expression is particularly important during the clearance of apoptotic cells and other cellular debris, as it would decrease the potential for generating an aberrant immune response against self proteins. Excess cytokine secretion would otherwise lead to chronic inflammatory processes and complement-mediated tissue damage. MBL modulates cytokine production at both the mRNA and protein levels. It contributes signals to peripheral blood monocytes, leading to the suppression of LPS-induced proinflammatory cytokines, IL-1α and IL-1β and an increase in the secretion of anti-inflammatory cytokines IL-10, IL-1 receptor antagonist, monocyte chemotactic protein 1 (MCP-1) and IL-6 [103,104]. Jack et al., using Neisseria meningitidis incubated with increasing concentrations of MBL before being added to MBL-deficient whole blood, observed that the release of tumour necrosis factor α (TNF-α), IL-1β and IL-6 from monocytes was enhanced at MBL concentrations below 400 ng/ml but suppressed at higher concentrations [105]. MBL concentrations were also inversely correlated with IL-1β, IL-12, granulocyte/monocyte-colony stimulating factor (GM-CSF), TNF-α and other cytokine and chemokine levels during acute malaria, which may be related to the level of MBL-mediated phagocytosis of infected erythrocytes and hemozoin [106]. Furthermore, ligand-bound MBL stimulates polymorphonuclear leukocytes to induce cell aggregation and superoxide production [107].

2d. Recognition of Altered Self and Apoptosis A role for MBL in the clearance of apoptotic cells was first proposed by Ogden et al. in 2001 [108]. MBL was found to recognize ACAMPs and bind directly to apoptotic cells that expose terminal sugars of cytoskeletal proteins, thereby permitting their recognition and directly facilitating their phagocytosis by macrophages [109]. C1q and MBL have also been shown to facilitate binding of apoptotic cells to immature dendritic cells [110]. Defects in the clearance of apoptotic cells have been implicated in the pathogenesis of certain autoimmune conditions, although the precise role of MBL, if any, remains unknown. For example, in 2005, Stuart et al. reported that although MBL-deficient mice displayed defective apoptotic cell clearance, they did not develop autoimmune diseases [111]. In animal studies, MBL has been implicated in the pathophysiology of ischaemia/reperfusion injury due to its ability to recognize altered self-structures.

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Introduction

15

The lectin pathway is a mediator of this process in certain organs, and the absence of MBL/MASP pathway activation appears to afford protection in these disease models [112,113]. Furthermore, changes in cell surface structures during oncogenic transformation appear to promote binding of MBL to cancer cells [114] where the protein can mediate cytotoxic effects including MBL-dependent cell mediated cytotoxicity [115,116]. The relative importance of such mechanisms in tumour immunology is, at present, unknown. In B cell follicular lymphoma, which represents approximately 40% of all non-Hodgkin lymphomas, there is a high proportion of immunoglobulin (Ig) variable region N-linked glycosylation sites (in 79% of patients compared to 9.3% of the normal population) that become occupied by oligomannose structures [117,118]. MBL has been demonstrated to bind both purified follicular lymphoma IgG, and cells from a B lymphoma cell line which express a cell surface IgM carrying such a variable region glycosylation site [119]. This may implicate MBL in B cell activation and proliferation, however, therapeutically, MBL could serve as carrier of chemotherapeutic drugs to these cells.

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2e. Other MBL Functions There are endogenous serum proteins which naturally carry appropriate glycans for MBL to bind to, such as the thiol-ester protein family (complement C3 and C4, and α-2 macroglobulin) [120]. MBL can further interact with certain Igs, generating cross communication between the innate (MBL) and adaptive (Ig) immune system. MBL binds agalactosylated glycoforms of IgG (IgG-G0), polymeric forms of IgA and certain glycoforms of IgM which have a high content of GlcNAc-terminating glycans. This interaction provides a route for clearance of immune complexes from the serum, and a mechanism of complement activation to Ig-coated pathogens [121]. There also seems to exist an alternative function for MBL with autologous cells. In this respect, it has been observed that most human monocytes and monocyte-derived dendritic cells contain intracellular MBL and that MBL binds through its CRD with a ligand/receptor on dendritic cells [122]. MBL can also bind to B cells, but probably only at the high concentrations reached at inflammatory sites, where a local elevation of extravascular MBL concentration and an increased influx of these cells are to be expected [123]. The functional implications of these recent-discovered links between the innate and adaptative immune system are still under investigation. There is also a link between the lectin pathway and the coagulation system. MASP-1 has thrombin-like activity, and activates factor XIII and cleaves

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Iara de Messias-Reason and Angelica B. W. Boldt

fibrinogen directly [124]. When MBL or ficolins, with associated MASP-1, 2 and 3 bind to targets, both MASP-1 and MASP-2 may contribute to localized fibrinogen activation. MASP-2 has a low activation potential generating only a limited amount of thrombin, which will be active for a short time due to inhibitors found in plasma. Therefore, fibrin will probably be deposited on the surface to which MASP-2 is bound, attracting phagocytes and serving as adhesion points for these immune cells [125].

3. MBL2 GENE POLYMORPHISM There are at least 87 polymorphic sites in a 10 kb region that includes the MBL2 gene [126]. The overall variation observed in the MBL2 genomic region of 1166 chromosomes from 24 different ethnic groups actually resulted in levels of nucleotide diversity (π=22.3 x 10-4) [127], which are three times the average estimate for the human genome (π=7.51 x 10-4), exceeding the upper limit of the 95% interval defined by the SNP consortium (π = 2 x 10-4 - 15.8 x 10-4) [128].

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3a. MBL2 Variants and Allelic Haplotypes Both circulating levels of MBL oligomers and functional activity have been correlated with common MBL2 genetic variants. There are at least 28 segregating sites in the MBL2 promoter and exon 1 sequence, and 21 allelic haplotypes were already physically defined (Figure 4, Table 2) [126,129,106,130,131,132,133,127]. Exon 1 harbours at least one synonimous (codon 44 for asparagine [130]) and eight non-synonimous variants. Rare substitutions in African haplotypes exchange leucine by glycine in codon 12 (Leu12Gly, allele F); cysteine by serine in codon 25 (Cys25Ser, allele E), serine by asparagine in codon 39 (Ser39Asn), serine by alanine in codon 40 (Ser40Ala) and in codon 52, arginine by histidine (Arg52His) [106,133,134]. Among the frequent variations, substitutions in codon 52 (CGT to TGT) exchanges arginine with a cysteine (Arg52Cys, allele D); in codon 54 the changing of GGC to GAC causes the substitution of glycine with aspartic acid (Gly54Asp, allele B), and in codon 57 the change of GGA to GAA causes the substitution of glycine with glutamic acid (Gly57Glu, allele C) [106,135,136]. These mutations have a profound effect on the assembly and stability of the protein, which leads to an increase of low-molecular-mass MBL that has reduced

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Introduction

17

capacity of activating complement and of ligand binding [137,21] and therefore to functional MBL deficiency in homozygous (e.g., B/B) or compound homozygous (e.g., B/C) carriers. Individuals with the B and C structural gene mutations express an unstable protein due to the interruption of the Gly-Xaa-Yaa repeat motif of the collagenous domain, reducing molecular stabilisation through normal disulphide linkages [138,139]. Heise et al. (2000) also found that formation of the B and C variant 32 kDa MBL structural subunit is hindered due to a failure of glycosylation of neighbouring lysine residues (Figure 2) [57]. The D mutation interferes to a lesser degree with the collagen structure of MBL than the B and C mutations. The common D, B and C SNPs have been collectively labeled O, whereas the major alleles at these loci have been called A. Whereas O/O individuals have near undetectable levels of high-order MBL oligomers, A/O individuals may have a 2- to 100-fold reduction [140,135,141,136,132,142,138]. Apart from oligomerization defects of the structural subunit, effective matrix metalloproteinase (MMP) cleavage of mutant MBL but resistance of normal MBL provides an explanation for the absence of MBL in O/O homozygotes but persistence of low levels of MBL in A/O heterozygotes. Recombinant rat MBL proteins with mutations homologous to those found in human variant MBL were rapidly and concentration-dependently cleaved at specific sites by the gelatinolytic MMPs, MMP-2, MMP-9, and the MMP-2 activator MMP-14, probably due to the exposure of cleavage sites within the perturbed triple helix [143] (Figure 2). Previous studies have indicated that MBL in at least a tetrameric form is required to enable activation of the complement pathway, whereas MBL dimers or trimers may opsonise pathogens sufficiently for phagocytic recognition and processing, although with a profound reduced avidity compared to high-order oligomerized MBL [137,144,25]. Since structural units comprised of two or three variant subunit chains are not able to bind MASP and activate the lectin pathway of complement, MBL tetramers composed of more than two of these will also be dysfunctional [75]. Accordingly, circulating high order oligomeric MBL of heterozygous A/B individuals do not correlate with normal mannan binding levels and C4 deposition capacity [145]. In addition, at least three SNPs in the promoter region modulate the concentration of the protein in serum: MBL2*H,L (located 550 bp before the transcription start site), X,Y (located 221 bp before the transcription start site) and P,Q (not coding SNP located 4 bp after the transcription start site) [136,132]. The combination of structural gene and promoter polymorphisms results in a dramatic variation in MBL concentration in apparently healthy individuals of up to 1000fold (European: range A → p.Gly54Asp g.273C>G

MBL2*HYPD (Y16582)

g.[273C>G; 1045C>T]→ p.Arg52Cys

MBL2*LXPA (Y16580)

g.602G>C

MBL2*LYQA (Y16576)

g.[396>C; 474A>G; 487A>G; 495_500del6; 753C>T; 826C>T]

MBL2*LYQC (Y16578)

g.[396>C; 474A>G; 487A>G; 495_500del6; 753C>T; 826C>T; 1061G>A]→ p.Gly57Glu g.1045C>T→ p.Arg52Cys g.487A>G

MBL2*LYPD similar to MBL2*LYPA similar to MBL2*LYQA Absolute Linkage Disequilibrium (ALD) with MBL2*LYPA

(MBL2*LYPF) ALD with MBL2*LYPB ALD with MBL2*LYQA (MBL2*LYQE) ALD with MBL2*LYQC

g.[474>G; 487A>G; 495_500del6; 753C>T; 826C>T] g.259C>T g.388G>A g.[388G>A; 477C>T] g.478G>A g.658C>A g.[925C>G; 926T>G]→ p.Leu12Gly g.788T>C g.578G>A g.659C>T g.965G>C → p.Cys25Ser g.[797C>A] g.[482A>G; 797C>A] g.[712A>T]

Common in all populations exceptig Amerindians and Aboriginals (~ 15 20%) Common in all populations excepting Amerindians and Aboriginals (~ 15 25%) Common in African populations (~ 15%) ~ 1% in European populations Rare ancestral allelic haplotype Rare ancestral allelic haplotype ~ 2% in African populations Common in African, Asian and Amerindian populations (~ 6 - 15%) Rare African haplotype Rare African haplotype Rare African haplotype Rare African haplotype ~ 2% in the Kaingang Amerindian ~ 5% in African populations Rare African haplotype Rare African haplotype ~ 2% in African populations Rare African haplotype ~ 2% in African populations

Reference sequence: Y16577, NP_000233. Amino acid changes were deduced from sequence variations in bold. Their description is preceded by an arrow. Mannose-Binding Lectin in the Innate Immune System, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook

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Introduction

19

Linkage disequilibrium between the SNPs in the promoter region and exon 1 is responsible for the occurrence of only seven common haplotypes (as opposed to the 64 theoretically possible) in African, European, Asian, Aboriginal and Amerindian populations, which are associated with progressively lower MBL serum concentration: MBL2*HYPA > LYQA > LYPA > LXPA >> HYPD = LYPB = LYQC (Table 3) [106,51,26,136,132,142,146]. The LYQA and LYQC haplotypes are further defined by the g.396A4C, g.474A4G, g.487A4G, g.495_500del6, g.753C4T and g.826C4T segregating sites, and there are ancient rare ‘intermediate’ haplotypes between LYQA and LYPA (LYQA without the g.396A4C variant, and LYPA with g.487A4G) (Table 2) [106]. LYPD also occurs at around 1% frequency in European-derived populations and was probably generated by a recent intragenic recombination event between the LYPA and HYPD or the LYPB and HYPD haplotypes in the European population [147,148,149]. The importance of the LX promoter is highlighted in the A/D individuals, since LXA/HYD individuals demonstrate reduced high molecular weight oligomers and severely reduced mannan binding and C4 deposition, in contrast to HYA or LYA /HYD individuals [145]. Estimates of linkage disequilibrium across MBL2 gene indicate that it is divided into two blocks, with comparable density of common SNPs and a probable recombination hot spot in the 3’ end [126,150]. There are another 25 SNPs within this recombination hot spot in exon 4, most of them in the UT-coding region. Among those in the CRD-coding region, two are synonimous (leucine at codon 126 and asparagine at codon 136) and two, non-synonimous: a change from asparagine to serine at codon 176 (Asn176Ser) and a change from glutamic acid to a premature stop codon at codon 209 (Glu209UAG) (Figure 2), with 2% frequencies in Hispanic and African populations, respectively [126]. The 3130G>C SNP that does not modify the coding information for leucine at position 126 influence functional MBL levels within the LXPA haplotype in the European population [151]. LXPA carriers with 3130G showed a significantly lower geometric mean functional MBL serum level of 190 ng/mL compared with 700 ng/mL in 3130C carriers This explains why European LXPA homozygotes display a much larger variation in MBL levels than homozygotes for the other common haplotypes (HYPA, LYPA or LYQA). The 3130G>C SNP is in linkage disequilibrium with the intronic IVS31709G>C SNP, which might be actually responsible for the observed phenotype [151]. Additional 5' variants as well as markers of distinct 3' haplotype blocks in MBL2 gene may also contribute to circulating protein levels [150].

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Iara de Messias-Reason and Angelica B. W. Boldt

Table 3. Average high-order oligomeric MBL concentrations in serum according to the most frequent genotypes in four populations: Danish and Australians of European origin, Mozambican Africans, Koreans, SouthAmerindian Chriguanos and Eskimos [26,136,132,142,263,33] MBL2 genotype HYA/LYA

[MBL] (ng/ml) 1887 3650

n

Africans

50

[MBL] (ng/ml) nt

n

Koreans

---

[MBL] (ng/ml) 2303 2750

Chiriguanos n 21

[MBL] (ng/ml) nt

n ---

Eskimo [MBL] (ng/ml) nt

n ---

LYA/LYA

1897 3710

21

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