El reajuste o retiro 25.1 El retiro

The second group of vertebrate CTLDcps is large and heterogeneous. It can be divided into four subgroups based on the gene linkage in mammals: asialoglycoprotein receptor (ASGR or ASGPR) gene cluster (ASGR1, ASGR2, HML, MMGL), dendritic cell (DC) receptor gene cluster (DC-SIGN paralogues and CD23), macrophage receptor gene cluster (Dectin-2, Mincle, DCIR, DLEC/BDCA-2) and several lone members. A more conventional approach to discussion of most of the group II members found in the literature is based on grouping by expression profile and function (macrophage

receptors, dendritic cell receptors etc.`; Figdor et al. 2002; Geijtenbeek et al. 2004), but

this is often not consistent with phylogeny, and brings members of different groups into one set.

As for the group V CTLDcps, members of group II are type II transmembrane proteins, which contain a short cytoplasmic tail, a transmembrane domain, a stalk

Chapter 1 Introduction region and an extracellular domain. The length of the stalk region, which is involved in oligomerization, varies between different members of the group. Most of the group members have an established carbohydrate-binding activity, although the functional importance of carbohydrate binding varies.

I will describe the group in detail, as it has significantly expanded recently, and because to the best of my knowledge it has not been reviewed from the point of view of CTLD evolution.

Asialoglycoprotein receptor subgroup

The asialoglycoprotein receptors subgroup includes two members (ASGR1 and ASGR2) found in different mammals, and one and two more members identified in human and rodents, respectively. In sequenced mammalian genomes the genes encoding proteins from this subgroup are clustered. Asialoglycoprotein receptor (ASGR or

ASGPR`; reviewed in Schwartz 1984; Stockert 1995), also known as hepatic lectin, was one of the first C-type lectins discovered (Stockert et al. 1974; Kilpatrick 2002). The

receptor functions as a heterotrimer made of two distinct subunits (ASGR1 and ASGR2 - major and minor, respectively). In rat and mouse ASGR2 is found in two differently glycosylated forms, which were initially considered as separate proteins (rat hepatic

lectin (RHL) 2 and 3 or RHL2/3`; Hong et al. 1988). Heterooligomeric structure is

essential for high-affinity binding and internalization (McPhaul and Berg 1986; Braun

et al. 1996). Interestingly, rat spermatogenic cells express an unusual oligomer of

ASGR2 called rat sperm galactosyl receptor, that consists of a full-length form and a truncated form that lacks the C-terminal part of the CTLD (Abdullah and Kierszenbaum 1989; Rivkin et al. 2000).

ASGR binds and internalizes galactose-terminated oligosaccharides of desialylated glycoproteins. After the ligand dissociates in the acidic environment of lysosomes, ASGR is recycled to the cell surface. The mechanism of the galactose binding has many interesting aspects, as the subunits have different monosaccharide specificity (Ruiz and Drickamer 1996), and bind the same complex carbohydrate molecule simultaneously, which is unusual for CTLDcps.

Chapter 1 Introduction A protein called “chicken hepatic lectin” was isolated and sequenced shortly after the mammalian ASGRs (Drickamer 1981). However, sequence analysis now shows that it is more similar to proteins from the DC-SIGN subgroup, which is consistent with its specificity for mannose-type ligands (N-acetyl-D-glucosamine, Kawasaki and Ashwell 1977; Loeb and Drickamer 1987).

Unlike ASGRs, which are found exclusively on liver parenchyma, other members of the ASGR gene cluster are expressed by macrophages. The macrophage galactose- binding lectins1 (MGL) are present in two copies in mouse (mMGL1, Ii et al. 1990; and

mMGL2, Tsuiji et al. 2002), but only one copy is found in human (hMGL, also called

human macrophage lectin, HML`; Suzuki et al. 1996). Recombinant mMGL1 and

mMGL2 CTLDs have shown differences in carbohydrate-binding specificities (Tsuiji et

al. 2002).

DC-SIGNs and CD23

Dendritic cell-specific ICAM-grabbing non-integrin (DC-SIGN, CD209) and its close homologue DC-SIGNR (DC-SIGN receptor) have recently become the most topical of the C-type lectins, because of their binding of the GP120 protein from the human immunodeficiency virus (HIV) envelope. Internalization of HIV by DC-SIGN (Engering et al. 2002) is responsible for viral particle transfer and in-trans infection of

susceptible T-cells. Apart from HIV, DC-SIGN is a receptor for a number of other pathogens:Mycobacterium tuberculosis (Geijtenbeek et al. 2003), hepatitis C virus

(Wang et al. 2004), Ebola virus (Alvarez et al. 2002), and human cytomegalovirus

(Halary et al. 2002).

The DC-SIGN subgroup is an actively evolving gene family, and significant differences are observed between mammals. Two genes (DC-SIGN and DC-SIGNR) were identified in human, a group of paralogues was found in nonhuman primates

(Bashirova et al. 2003), and five DC-SIGN homologues were found in mouse (Park et

al. 2001), which were named DC-SIGN2, SIGNR1, SIGNR2, SIGNR3 and SIGNR4.

1 _{Not to be confused with the macrophage C-type lectin (MCL).}

2 _{mDC-SIGN was identified as the hDC-SIGN orthologue based on the proximity of its gene to the}

Chapter 1 Introduction Interestingly, in the fish genome the DC-SIGN group is also expanded (Chapter 5). Two works describing structural studies of DC-SIGN(R) complexed with oligosaccharides have been published recently (Feinberg et al. 2001; Guo et al. 2004). Carbohydrate

recognition plays a central role in the DC-SIGN binding of pathogens.

CD23 (reviewed by Bonnefoy et al. 1997; Mossalayi et al. 1997; Kijimoto-Ochiai

2002), also known as the low affinity IgE receptor (FcIgE2R) is a key molecule of B- cell activation and growth (regulation of IgE synthesis). It is a glycoprotein expressed on a number of cell types including lymphocytes, eosinophils, platelets, and

macrophages, and is also found in a soluble form, which is produced by proteolysis. The stalk region of CD23 has D-helical structure and is involved in oligomerization (tri- or tetra-) of the receptor, by coiled-coil formation (Beavil et al. 1992), which

significantly increases its affinity for IgE (Dierks et al. 1993). The CTLD of CD23 is

involved in both protein-protein and protein-carbohydrate interactions. Although human

CD23 binds IgE in a carbohydrate-independent manner (Vercelli et al. 1989),

recognition of another ligand (CD21) and CD23-induced cell aggregation require Gal-

terminated glycan chains (Kijimoto-Ochiai and Uede 1995; Kijimoto-Ochiai et al.

1994; Aubry et al. 1994). The predicted Ca2+-binding site 2 motifs of human CD23

CTLD are EPT and WND (EPN in mouse, rat and horse), which are typical for mannose-binding CTLDs, so galactose specificity of CD23 is unexpected.

Recently a new CTLDcp encoded by the DC-SIGN gene cluster has been characterized (Liu et al. 2004). The protein (LSECtin) was found in sinusoidal

endothelial cells of human liver and lymph node, which is similar to the expression profile of DC-SIGNR. The CTLD contains an EPN motif and, as expected,

preferentially binds mannose-type ligands.

Macrophage receptors

The macrophage receptor cluster (Flornes et al. 2004; Balch et al. 2002) is located

on chromosome 12p13 in human (Ch 6F2 in mouse) and is closely linked to the NK cell receptor complex (group V, see p. 29). The cluster encodes several CTLDcps expressed

by macrophages and dendritic cells: macrophage C-type lectin (MCL`; Balch et al.

Chapter 1 Introduction

cell immunoreceptor (DCIR`; Bates et al. 1999), dendritic cell lectin (DLEC or BDCA-

2`; Arce et al. 2001; Dzionek et al. 2001) and dendritic cell-associated lectin-2 (Dectin-

2`; Ariizumi et al. 2000a; Kanazawa et al. 2004). The rodent gene cluster contains

several members that are not present in human, such as DCIR paralogues (DCIR2-

DCIR4, Flornes et al. 2004), and a dendritic cell immunoactivating receptor (DCAR`;

Kanazawa et al. 2003). The members of this subgroup were discovered relatively

recently, and their function is poorly understood (Ariizumi et al. 2000a). To my

knowledge, the only information about carbohydrate-binding properties of these proteins comes from two studies on Dectin-2, which gave conflicting results: in one

case Ca2+-dependent binding to mannose was observed (Fernandes et al. 1999), while in

the other the protein did not bind carbohydrate (Ariizumi et al. 2000a). In all group

members, a Ca2+-binding site 2 motif is present, although in some cases this has an unusual sequence (e.g. EPK, ESN, EPD in rat, mouse and human MCL, respectively).

Langerin and Kupffer Cell Receptor

A cluster of two genes on human chromosome 2p13 (mouse Ch 6D1) encodes CTLDcps Langerin (CD207) and Kupffer cell receptor (KuCR). Langerin is an

endocytic receptor uniquely expressed by Langerhans cells and associated with Birbeck granules (human, Valladeau et al. 2000; mouse, Takahara et al. 2002). It has a long

stalk region that is involved in trimerization of the receptor by coiled-coil formation. The CTLD of Langerin has typical motifs associated with mannose binding, and it has been shown that the protein indeed binds mannose-group monosaccharides (Stambach and Taylor 2003). Kupffer cell receptor from rat has a structure similar to Langerin, but is expressed in liver and functions as an endocytic receptor for fucose-terminated glycoproteins (Hoyle and Hill 1988; Lehrman and Hill 1986). The KuCR locus in the human genome lacks the 3’-terminal exon, which truncates the CTLD at the beginning of the LLR; this led Fadden and coworkers to suggest that the human receptor is a pseudogene, which was also supported by their failure to identify hKuCR cDNA

expression by PCR or by EST database search (Fadden et al. 2003). However, full-

length cDNA (AK096429) for hKuCR is now available in GenBank. Also, an alignment between human and rat proteins shows a high level (63% identity) of conservation. Rat

Chapter 1 Introduction KuCR, which contains a galactose-type QPD motif is also interesting as it binds fucose with a relatively high affinity (Fadden et al. 2003).

Other group II members

Scavenger receptor with a C-type lectin domain (SRCL, human Ch 18.p11`; Nakamura et al. 2001a; Nakamura et al. 2001b) has an unusual structure for group II

proteins. Its long stalk region contains a collagen domain between the juxtamembrane coiled-coil region and the CTLD: this is why it is also described as a placental collectin

(CL-P1`; Ohtani et al. 2001) and its HUGO-approved name is COLEC12. However,

despite the presence of the collagen region, the domain structure of the protein is analogous to other group II CTLDcps, and phylogenetic analysis of the CTLD alignments confidently places SRCL into the group II branch. The CTLD of SRCL is similar to the ASGR CTLDs and includes all the elements which were shown to contribute to high-affinity galactose binding by ASGR (QPD motif, a tryptophan and glycine-rich loop`; Kolatkar and Weis 1996). As expected, SRCL binds galactose-type ligands (Yoshida et al. 2003).