G. S. Gupta

Chi-Lectins: Forms, Functions and Clinical Applications

Chitinases (EC.3.2.1.14) hydrolyze the β-1,4-linkages in chitin, an abundant N-acetylglucosamine (GlcNAc) polysaccharide that is a structural component of protective biological matrices of invertebrate such as insect exoskeletons and fungal cell walls. Chitinases cleave chitin and contain the conserved sequence motif DXXDXDXE, in which the glutamate is the catalytic residue. Chitinases are found in species including archaea, bacteria, fungi, plants, and animals. On the basis of sequence homologies, chitinases fall into two groups: families 18 and 19 of glycosyl hydrolases. Members of family 18 employ a substrate-assisted reaction mechanism (van Aalten et al. 2001), whereas those of family 19 adopt a fold-and-reaction mechanism similar to that of lysozyme (Monzingo et al. 1996), suggesting that these families evolved independently to deal with chitin. The glycoside hydrolase 18 (GH18) family of chitinases is an ancient gene family widely expressed in archea, prokaryotes and eukaryotes. Since chitin is an important structural component of pathogens like fungi as well as a constituent of the mammalian diet, a dual function for mammalian chitinases in innate immunity and food digestion has been envisioned (Suzuki et al. 2002; Boot et al. 2005a). Indeed, for human chitotriosidase, an enzyme predominantly expressed by phagocytes, a fungistatic effect has been demonstrated (van Eijk et al. 2005). Several studies have tried to link a common chitotriosidase deficiency to susceptibility for infection by chitin-containing parasites (Bussink et al. 2006). The physiological function of the second mammalian chitinase, acidic mammalian chitinase (AMCase), has attracted considerable attention due to a report linking the protein to the pathophysiology of asthma (Zhu et al. 2004).

Assessing the existing learning methodology in physiology: a feedback study from students of two medical colleges in Northern India

To improve the overall health scenario of the nation, the need of the hour is to produce quality doctors and not just the quantity. To achieve this, medical education needs to be updated keeping in mind the current trends. Physiology, one of the basic subjects of medical education forms the foundation of many medical branches and students should have its proper understanding. The aim of the study was to assess the current practices and attitudes of the medical students in two medical colleges of North India in the Jammu Province of J&K towards learning of physiology. 500 medical students participated in this study, 300 from the ASCOMS & Hospital, Sidhra, Jammu and 200 from the Government Medical College(GMC), Jammu. A questionnaire consisting of 13 questions was provided to each of them to elicit their feedback regarding learning of physiology. The results revealed that 89% were in favour of lectures, 51.2% of tutorials and 83.45% were of the opinion that practicals were useful. Enthusiasm of the students ’in learning physiology was so much that 36.2% considered lecture notes insufficient, so 87.4% were using advised reference books, 51.2% previous examinations and 57.6% previous exam papers to guide them. When faced with difficulty in learning physiology majority preferred to consult advised reference book, class fellows and seniors in that order, while consulting teachers was found to be the last option. The potential to self-study was so much that 64.8% used internet and 21% scientific journals to get the latest information in the learning of the subject. We can conclude that medical students are motivated in learning physiology by using hybrid techniques which include using advised reference books, consulting among themselves to solve problems and using online sources Fewer consultation with teachers and lesser interest in tutorials can be improved by initiative from teachers, by becoming more friendly with students, encouraging them, creating an open and positive atmosphere, being more responsive and empathetic to them.

R-Type Lectin Families

The R-type lectins are members of a superfamily of proteins, all of which contain a carbohydrate-recognition domain (CRD) that is structurally similar to the CRD in Ricin. The R-type domain is an ancient type of protein fold that is found in many glycosyltransferases as well as in bacterial and fungal hydrolases. Interestingly, the R-type CRD is the only one conserved between animal and bacterial lectins (Sharon and Lis 2004). Ricin was the first lectin discovered and it is the prototypical lectin in this category. Two different lectins have been purified from R. communis seeds, and in the original nomenclature they were termed RCA-I and RCA-II. RCA-I is an agglutinin but a very weak toxin. RCA-II is commonly called ricin, and it is both an agglutinin and a very potent toxin. The designation RCA-II has now been dropped, but the original name for the agglutinin RCA-I has been retained. The molecular mass of RCA-I is approximately 120 kDa and that of ricin is approximately 60-kDa. Ricin is a type II ribosome-inactivating protein (RIP-II). Although one might predict that RCA-I also would be highly toxic, it has weak activity compared to ricin because it lacks a separate A chain (Fig. 14.1).

Mannose Receptor Family: R-Type Lectins

R-type lectins exist ubiquitously in nature and mainly bind to galactose unit of sugar chains. Originally found in plant lectin, Ricin, the R-type lectin domain is found in several animal lectins, including the members of mannose receptor (MR) family, and in some invertebrate lectins (discussed in Chap. 14). The R-type domain contained in these proteins is the CRD, which is also termed a carbohydrate-binding module (CBM) and has been placed in the CBM13 family in the CAZy database (carbohydrate-active enzymes database). While the A chain in ricin has eight α-helices and eight β-strands, and is the catalytic subunit, the B chain contains R-type lectin domains, has two tandem CRDs that are about 35 A apart and have a shape resembling a barbell, with one binding domain at each end. Each R-type domain has a three-lobed organization that is a β-trefoil structure (from the Latin trifolium meaning “three-leaved plant”). The β-trefoil structure probably arose evolutionarily through gene fusion events linking a 42-amino-acid peptide subdomain that has galactose-binding activity. The three lobes are termed α, β, and γ and are arranged around a threefold axis. Conceivably, each lobe could be an independent binding site, but in most R-type lectins only one or two of these lobes retain the conserved amino acids required for sugar binding. The R-type domain is also found in pierisin-1, which is a cytotoxic protein from the cabbage butterfly Pieris rapae, and in the homologous protein pierisin-2, from Pieris brassicae. Tandem R-type motifs are found in some other R-type family members. For example, Limulus horseshoe crab coagulation factor G has a central R-type lectin domain, which is flanked at the amino terminus by a xylanase Z-like domain and at the carboxyl terminus by a glucanase-like domain. In this chapter we will restrict our discussion to R-type lectins of mannose receptor family, which comprises also of endocytic receptors.

KLRB Receptor Family and Human Early Activation Antigen (CD69)

Natural killer cells are important component of the innate immune system, providing protection against intracellular infection particularly viruses and also neoplasia through direct cytotoxic mechanisms and the secretion of cytokines. They mediate their effects through direct cytolysis, release of cytokines and regulation of subsequent adaptive immune responses. They are called ‘natural’ killers because, unlike cytotoxic T cells, they do not require a previous challenge and preactivation to become active. NK cells can be activated by a range of soluble factors, including type I interferons, IL-2, IL-12, IL-15 and IL-18, but also by direct cell to cell contact between NK cell receptors and target cell ligands. NK cells possess an elaborate array of receptors, which regulate NK cytotoxic and secretory functions upon interaction with target cell MHC class I proteins. Determination of structures of NK cell receptors and their ligand complexes has led to a fast growth in our understanding of the activation and ligand recognition by these receptors as well as their function in innate immunity. B and T cells significantly and differentially influence the homeostasis and the phenotype of NK cells. The function of NK cell is tightly regulated by a fine balance of inhibitory and activating signals that are delivered by a diverse array of cell surface receptors. A prerequisite for a NK cell attack is the presence on target cells of ligands for activating receptors and low level or absence of ligands for inhibitory receptors. It was believed that NK self-tolerance was achieved by expression on each NK cell of at least one self-MHC specific inhibitory receptor. However, this dogma has been challenged after identification of a NK cell population in normal mice that lack inhibitory receptors specific for self-MHC class I molecules (Kumar and McNerney 2005; Fernandez et al. 2005). Therefore, it was made clear that some additional surface receptors contribute to NK self-tolerance and to the modulation of NK cell responses. The characterization and the identification of their physiological ligands allow us a comprehensive understanding of NK cell function.

Dectin-1 Receptor Family

Natural killer (NK) cell receptors belong to two unrelated, but functionally analogous gene families: the immunoglobulin superfamily, situated in the leukocyte receptor complex (LRC) and the C-type lectin receptors (CLRs) superfamily, located in the natural killer gene complex (NKC). Wong et al. (2009) described the largest NK receptor gene expansion seen to date and identified 213 putative C-type lectin NK receptor homologs in the genome of the platypus. Many have arisen as the result of a lineage-specific expansion. Orthologs of OLR1, CD69, KLRE, CLEC12B, and CLEC16p genes were also identified. The NKC is split into at least two regions of the genome: 34 genes map to chromosome 7, two map to a small autosome, and the remainder are unanchored in the current genome assembly. No NK receptor genes from the LRC were identified. The massive C-type lectin expansion and lack of Ig-domain-containing NK receptors represents the most extreme polarization of NK receptors found to date. This new data from platypus was utilized to trace the possible evolutionary history of the NK receptor clusters.

NKG2 Subfamily C (KLRC)

NKG2 receptors are type II C-type, lectin-like, integral membrane glycoproteins, which are expressed on the cell surface as heterodimers with CD94, which is an invariant type II C-type, lectin-like polypeptide. CD94 lacks a cytoplasmic tail and therefore, cannot transduce signals. It is however essential for the expression of NKG2 receptors. Four distinct genes, A/B, C, E/H, and F, encode the NKG2 receptors. Of these receptors, CD94/NKG2A is an inhibitory one, as it contains a long cytoplasmic tail with two ITIMs. Others have short cytoplasmic tails, and each associates noncovalently with a homodimer of DAP-12, as in the case of activating KIRs. The NKG2 family of genes (HGMW-approved symbol KLRC) contains at least six members (NKG2-A, -B, -C, -E, -F and -H) which are localized to human chromosome 12p12.3-p13.2, in the same region where CD69 genes have been mapped. In addition, the human CD94 and NKR-P1A genes map to the short arm of chromosome 12. The physical distance spanned by NK gene complex (NKC) in humans ranges between 0.7 and 2.4 megabases (Renedo et al. 1997). The NKG2 and CD94 genes are localized in a small region (< 350 kb) and mapped in the following order: (NKG2-C/NKG2-A)/NKG2-E/NKG2-F/NKG2-D/CD94. Sequence analysis of 62 kb spanning the NKG2-A, -E, -F, and -D loci allowed the identification of two LINE elements that could have been involved in the duplication of the NKG2 genes. Presence of one MIR and one L1ME2 element at homologous positions in the NKG2-A and NKG2-F genes is consistent with the existence of rodent NKG2 gene(s). The 5′-ends of the NKG2-A transcripts were mapped into two separate regions showing the existence of two separate transcriptional control regions upstream of the NKG2-A locus and defining putative promoter elements for these genes (Plougastel and Trowsdale 1998). Restriction mapping and sequencing revealed the NKG2-C, -D, -E, and -F genes to be closely linked to one another, and of the same transcriptional orientation. The NKG2-C, -E, and -F genes, despite being highly similar, are variable at their 3′ ends. It was found that NKG2-C consists of six exons, whereas NKG2-E has seven, and the splice acceptor site for the seventh exon occurs in an Alu repeat. NKG2-F consists of only four exons and part of exon IV is in some cases spliced to the 5′ end of the NKG2-D transcript. NKG2-D has only a low similarity to the other NKG2 genes Glienke et al. (1998). The murine NKG2-D-like sequence also maps to the murine NK complex near CD94 and Ly49 family members.

Overview of Animal Galectins: Proto-Type Subfamily

Lectins recognize and bind carbohydrates covalently linked to proteins and lipids on the cell surface and within the extracellular matrix, and they mediate many cellular functions ranging from cell adhesion to pathogen recognition. Phylogenetically conserved family of Galectins was defined in 1994 as a shared consensus of amino-acid-sequences of about 130 amino acids and the CRD responsible for s-galactoside binding (Barondes et al. 1994). The galectin (Gal) CRDs bind small β-galactosides/poly-N-acetyllactosamine-enriched glycoconjugates. But the overall binding affinity for more complex glycoconjugates varies substantially. To date, 15 members of the mammalian galectin family have been identified. Some, such as galectin-1, are isolated as dimers and have a single CRD in each monomer, whereas others, such as galectin-4, are isolated as monomers and have two CRDs in a single polypeptide chain. While CRDs of all galectins share an affinity for minimum saccharide ligand N-acetyllactosamine—a common disaccharide found on many cellular glycoproteins—individual galectins can also recognize different modifications to this minimum saccharide ligand and so demonstrate the fine specificity of certain galectins for tissue- or developmentally-specific ligands (Ahmad et al. 2004a). Location studies of galectins have established that these proteins can segregate into multiple cell compartments in function of the status of the cells in question (Danguy et al. 2002; Liu and Rabinovich 2005). Although galectins as a whole do not have the signal sequence required for protein secretion through usual secretory pathway, some galectins are secreted and are found in the extracellular space. While the intracellular activity of galectin-1 is mainly independent on its lectin activity, its extracellular activity is mainly dependent on it. The functions and distribution of Gal-1 and Gal-3 are well characterized.

NKG2D Activating Receptor

Information on receptor ligand systems used by NK cells to specifically detect transformed cells has been accumulating rapidly. Killer cell lectin-like receptor subfamily K, member 1, also known as KLRK1, is the product of human gene. The KLRK1 has been designated as CD314 and contains a C-type lectin-like domain (CTLD). KLRK1 is also known as: KLR; NKG2D; NKG2-D; FLJ17759; FLJ75772; D12S2489E. Human NKG2D was originally identified in 1991 as an orphan receptor on NK cells (Houchins et al. 1991). Although genetically mapping near the C-type lectin receptors CD94 and NKG2A-E, the NKG2D activating NK cell receptor has little sequence homology with these receptors and is expressed as a homodimer that signals through DAP10 rather than CD94 (Chap. 30). NKG2D binds to two distinct families of ligands, the MHC class I chain-related peptides (MICA and MICB) and the UL-16 binding proteins (ULBP). These ligands are upregulated in cells that have undergone neoplastic transformation, and NK cytotoxicity on tumor cells correlates with tumor expression of MICA and ULBP. The NKG2D differs from other members of the NKG2 family in significant ways. They do not form heterodimers with CD94 on the cell surface. Instead, they are expressed as homodimers, and each homodimer associates noncovalently with a homodimer of the adaptor protein DAP-10. The cytoplasmic tail of DAP-10 carries a YxxM motif, which can recruit the regulatory subunit p85 of phosphatidylinositol-3 kinase and Grb2 (see also Chap. 30).

DC-SIGN Family of Receptors

In the immune system, C-type lectins and CTLDs have been shown to act both as adhesion and as pathogen recognition receptors. The Dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) and its homologs in human and mouse represent an important C-type lectin family. DC-SIGN contains a lectin domain that recognizes in a Ca2+-dependent manner carbohydrates such as mannose-containing structures present on glycoproteins such as ICAM-2 and ICAM-3. DC-SIGN is a prototype C-type lectin organized in microdomains, which have their role as pathogen recognition receptors in sensing microbes. Although the integrin LFA-1 is a counter-receptor for both ICAM-2 and ICAM-3 on DC, DC-SIGN is the high affinity adhesion receptor for ICAM-2/-3. While cell–cell contact is a primary function of selectins, collectins are specialized in recognition of pathogens. Interestingly, DC-SIGN is a cell adhesion receptor as well as a pathogen recognition receptor. As adhesion receptor, DC-SIGN mediates the contact between dendritic cells (DCs) and T lymphocytes, by binding to ICAM-3, and mediates rolling of DCs on endothelium, by interacting with ICAM-2. As pathogen receptor, DC-SIGN recognizes a variety of microorganisms, including viruses, bacteria, fungi and several parasites (Cambi et al. 2005). The natural ligands of DC-SIGN consist of mannose oligosaccharides or fucose-containing Lewis-type determinants. In this chapter, we shall focus on the structure and functions of DC-SIGN and related CTLDs in the recognition of pathogens, the molecular and structural determinants that regulate the interaction with pathogen-associated molecular patterns. The heterogeneity of carbohydrate residues exposed on cellular proteins and pathogens regulates specific binding of DC-expressed C-type lectins that contribute to the diversity of immune responses created by DCs (van Kooyk et al. 2003a; Cambi et al. 2005).