ESSENTIALS OF GLYCOBIOLOGY LECTURE 22 MAY 4, 2004 Richard D. Cummings, Ph.D. University of Oklahoma Health Sciences Center College of Medicine Oklahoma.

ESSENTIALS OF GLYCOBIOLOGY

LECTURE 22

MAY 4, 2004

Richard D. Cummings, Ph.D.University of Oklahoma Health Sciences Center

College of MedicineOklahoma Center for Medical Glycobiology

“THE C-TYPE LECTINS AND THE SELECTINS”

Dr. Cummings

Historical Background Common Features of C-Type Lectins Localization and Functions of C-type Lectins The Endocytic Receptors The Collectins The Selectins The Natural Killer Lymphocyte Lectins Proteoglycans with C-type Lectin Domains Other Types of C-type Lectins

Outline

Dr. Cummings

Work initiated by Gilbert Ashwell and Morell 1960s and 1970s 125I-labeled sialylated glycoprotein given i.v. into rabbits

remained in serum with long half-life But the desialylated, 125I-labeled glycoprotein given i.v. was

rapidly removed from circulation Removal of terminal galactose from 125I-labeled desialylated

glycoproteins prolonged serum lifetime 125I-labeled desialylated glycoproteins sequestered in liver Ca2+-dependent receptor purified from rabbit liver membranes

Two subunits, trimeric structure Other related proteins purified: trimeric structures with

sequence similarities, but different carbohydrate binding specificity

Name C-type lectin family proposed by Drickamer in 1988, based on similarities in sequence and Ca2+-dependency

Prototypical Ca2+-dependent or C-type lectin: The Asialoglycoprotein Receptor (ASGPR)

Historical Background

Dr. Cummings

-ENWGAGEPNNKKSKEDCVEIYIKRERDSGKWNDDACHKRKAALCY-

-TNWNEGEPNNVGSGENCVVLLT-----NGKWNDVPCSDSFLVVCE-Mouse L-selectin

Rat Mannose Binding Protein C

-----------C---------------------------------C---------C-------C-------

LIVMSTA

FYWLIVSTA

—C— —X — —X— —X—X—C—X — — —C—

n = 5 to 12residues

DNSR

WL

LIVMFYATG

n m

m = 5 to 12residues

Conserved Carbohydrate-Recognition Domain of C-type Lectins

Common Cystine-structure in C-type Lectins

Common Features of C-type Lectins

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Common Features of C-type Lectins

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Superfamily of Proteins Containing a C-type Lectin Domain (CTLD)

Members of this superfamily share a common primary and secondary structure that coordinates Ca2+.

While all members of this superfamily of proteins have a CTLD, only about 1/2 of these members have a domain structure that promotes protein-carbohydrate interaction where the Ca2+is coordinated to the carbohydrate ligand.

The remaining members may use the CTLD to bind other types of ligands, e.g. polypeptides, and thus promote protein-protein interaction, as for lecticans (proteoglycans with a CTLD).

Common Features of C-type Lectins

Dr. Cummings

Ca2+

Ca2+ from another CRD

Ca2+-Coordination by the CRD of DC-SIGN

(From Feinberg et al, 2001)Man GlcNAc

2

23

6

1 2

3

45

Common Features of C-type Lectins

Dr. Cummings

Left: Structure of the CRD of DC-SIGN bound to GlcNAc2Man3

Right: Rat serum mannose-binding protein bound to a high-mannose oligosaccharide (From Feinberg et al, 2001)

Ca2+-Coordination by C-type Lectins

Common Features of C-type Lectins

Dr. Cummings

Crystal structure of a trimeric rat mannose-binding protein A (MBP-A) complexed with mannose at 1.8 Å resolution. Mannose is indicated in the stick figure and Ca2+ and Cl are indicated by the green and blue balls, respectively. Note that Ca2+ is closely coordinated with the bound sugar.(From Weis and Drickamer (1994) Structure 2:1227-40)

Common Features of C-type Lectins

Dr. Cummings

Localization and Function of C-type Lectins

The CTLD is a versatile domain found within many proteins, including enzymes, proteoglycans, and adhesion molecules.

C-type lectins are found as:

Secreted Proteins and Glycoproteins Transmembrane Proteins and Glycoproteins

(on both internal and plasma membranes)

The CTLD in C-type Lectins function in:

Cell-Cell Adhesion (e.g. Selectins) Glycoprotein Clearance by Endocytosis (e.g. ASGPR) Innate Immunity through Opsonizing Pathogens (e.g. Serum MBP) Innate Immunity through Endocytosis

and Antigen Presentation (e.g. DC-SIGN)and Co-Regulation with Toll Receptors

Dr. Cummings

Rat asialoglycoprotein receptor R2/3 (hepatic lectin 2/3) Human asialoglycoprotein receptor (hepatic lectin H1) Chicken hepatic lectin Rat kupffer cell receptor Human macrophage mannose receptor Dendritic cell and thymic epithelial cells DEC-205

(homolog of macrophage mannose receptor) Murine macrophage asialoglycoprotein-binding protein

(macrophage Gal/GalNAc-specific lectin - MMGL) Bovine 180 kD secretory phospholipase A2 receptor DEC 205 receptor Minkle (macrophage lectin inducible by TNF, IL-6 and INF-) DC-SIGN (Dendritic Cell-Specific ICAM-3 Grabbing Non-Integrin) the

related DC-SIGN(R) appear to mediate interaction of T cells with dendritic cells via recognition of glycans on ICAM-3

Endocytic Receptors

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Endocytic Receptors

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Endocytic Receptors

(From Geijtenbeek et al (2004)Ann. Rev. Immunol. 22, 33-54)

Dr. Cummings

“ Recognition of pathogens such as mycobacteria by the CLR DC-SIGN and TLRs can lead to immune activation when the TLR-signal overrules that of the CLR, and this includes DC differentiation. This can occur when low amounts of pathogens target the CLR. When high concentrations of pathogens target the CLR DC-SIGN, the immune tolerizing signals can overrule the TLR-induced signals and inhibit DC differentiation, leading to immune suppression and pathogen survival.” (From Geijtenbeek et al (2004)Ann. Rev. Immunol. 22, 33-54)

Toll Receptor

(TLR)

DC-SIGN

Endocytic Receptors

Dr. Cummings

Collectins (collagen-like sequences and lectin domains; function in innate immunity; fix complement in absence of antibody and have opsonin activity)

Human serum mannose-binding lectin (MBL) Rat mannose binding protein A and C Human pulmonary surfactant-associated protein A (SP-A) Human pulmonary surfactant-associated protein D (SP-D) CL-L1 CL-P1 Conglutinin (bovine) CL-43 (bovine) CL-46 (bovine), Ficolins

L-ficolin M-ficolin H-ficolin

Human tetranectin (TN) (Plasminogen-kringle 4 binding protein)

Collectins

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Collectins

Different Forms of Collectins

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mannan-binding lectin (MBL)(with attached GlcNAc)

Larger fgb of tachylectinfrom horseshoe crab

(with attached GlcNAc)

Tachylectinfrom horseshoe crab

(with attached GlcNAc)

Collectins

From: Holmskov et al (2003) Ann. Rev. Immunol. 21, 547-578

Crystal Structures of Collectins

Dr. Cummings

Selectins

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Similarities in C-type Lectin Domain Structureof Selectins and Other CTLDs

MBP-C E-Selectin P-Selectin

Selectins

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Selectins

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Selectins

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Selectins

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Selectins

Gal GalNAcGlcNAcFuc NeuAc S Sulfate

Ser/Thr

3

1

3

3

4

3

Core 2-based SIALYL LEWIS X EPITPOPE (sLex)

Dr. Cummings

Selectins

From: Somers et al (2000) Cell 103(3):467-79

From: Leppanen et al (2003) J Biol Chem 278(29):26391-400

Dr. Cummings

P-selectin Recognition of Fucose, Sialic, and Tyrosine Sulfate in

Glycosulfopeptides

Selectins

From: Somers et al (2000) Cell 103(3):467-79

Dr. Cummings

Gal GalNAcGlcNAcFuc NeuAc S Sulfate

Ser/Thr

3

3

1

3

3 4 3

4

3

6S

6S

6-sulfo-sLex

6-sulfo-sLex

MECA-79

Sulfated Glycans Recognized by L-selectin

Selectins

From: Rosen (2004) Annu Rev Immunol. 22:129-56.

Dr. Cummings

Lymphocyte lectins NK receptors (required for MHC-1 recognition)

(the CTLD functions in protein-protein interaction)Ly49A through WNKR-P1CD94/NKG2A/B, -C or -ENKG2DCD69

human mast cell function associated antigen (MCFA) activation-induced C-type lectin (AICL) human eosinophil granule major basic protein human low affinity IgE Fc receptor (CD23) (ON LYMPHOCYTES

AND MONOCYTES) P47 or LSLCL (lymphocytic secreted long form of C-type

lectin) CIRE (expressed by splenic dendritic cells)

The Natural Killer Lymphocyte Lectins

Dr. Cummings

Anatomy of C-type lectin-like domains of NK receptors. Ribbon diagrams of Ly49A, Ly49I (1JA3), NKG2D (1HQ8), CD69 (1FM5), CD94 (1B6E), and MBP-A. The secondary structural elements arecolored as follows: -strands blue, -helices red, and loop regions gold. The disulphide bonds are shown in green as ball-and-stick representation. The Ca2+ ions bound to MBP-A are drawn as magenta spheres.

Ly49A Ly49I

NKG2D CD69

CD94 MBP-A

Importance of the CTLDin Protein-Protein Interactions:

The NK Receptors

The Natural Killer Lymphocyte Lectins

Dr. Cummings

Recognition of MHC-I by the Ly49A, KIR2DL and NKG2D NK cell receptors. The KIR2DL2/HLA-Cw3 and NKG2D/MICA complexes (50, 58) were superimposed onto the Ly49A/H-2Dd complex using equivalent C atoms of the 1 and 2 domains of the MHC-I molecules. For clarity, the only MHC-I molecule shown is H-2Dd. The H-2Dd heavy chain is gold, the peptide is blue, and ß2m is gray. The Ly49A monomers interacting at Site 2 are cyan and light blue, KIR2DL is magenta, and the NKG2D monomers are green and pink. The overlapping area of KIR2DL and NKG2D is transparent.

From: Natarajan K, Dimasi N, Wang J, Mariuzza RA, Margulies DH. (2002) Annu Rev Immunol 20:853-85

Importance of the CTLDin Protein-Protein Interactions:

The NK Receptors

Dr. Cummings

human integral membrane protein DGCR2/IDD human lithostathine 1 aprecursor (pancreatic stone protein -

PSP) human polycystin human endothelial cell scavenger receptor human pancreatitis-associated protein 1 (PAP or HIP) human pancreatic beta cell growth factor (INGAP)

Proteoglycans human versican core protein (large fibroblasts

proteoglycan - CS proteoglycan core protein-2 - glial hyaluronate binding protein)

human aggregan core protein (cartilage-specific proteoglycan core protein - CSPCP- CS proteoglycan core protein-1)

rat brevican core protein (brain-enriched HA binding protein)

rat neurocan core protein (245 kD early post-natalcore glycoprotein)

Proteoglycans with C-type Lectin Domains

Other Types of C-type Lectins

Dr. Cummings

Invetebrate Lectins Limulus clotting factor (hemolymph of horseshoe crab

Tachypleus tridentatus) Lectin BRA-2 (coelomic fluid of acorn barnacle

Megabalanus rosa) Newt Lectin (oviduct of iberian ribbed newt Pleurodeles waltii) Inducible Flesh fly lectin (Sarcophaga peregrina) Tunicate lectin (Polyandrocarpa misakiensis) Integral Spicule matrix lectin in sea urchin

(Strongylocentrotus purpuratus) Sea urchin Echinoidin (Anthocidaris crassispina) Cockroach lectin (hemolymph of Periplaneta americana) Antifreeze protein (AFP) from the sea raven (Hemitripterus

americanus)

Viral Lectins Hepatic lectin homolog in Fowlpox virus gp22-24 in Vaccinia virus

Other Types of C-type Lectins

Dr. Cummings

Snakes and Venoms Alboaggregin A subunit 1 (white-lipped pit viper

(Trimeresurus albolabris) Phospholipase A2 inhibitor subunit B (Trimeresurus flavoviridis) Echicetin alpha subunit (saw-scaled viper Echis carinatus) Coagulation factor IX/factor X-binding protein A (IX/X-BP)

(Trimeresurus flavoviridis) Galactose-specific lectin (Crotalus atrox) Botrocetin, alpha chain (platelet coagglutinin) (Bothrops jararaca)

Other Types of C-type Lectins

Dr. Cummings