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An update on the the lectin pathway of complement

Peter Garred

Laboratory of Molecular Medicine

Department of Clinical Immunology, Section 7631

Rigshospitalet

Copenhagen

Denmark

E-mail: garred@post5.tele.dk

Malmø, September 17, 2012

Classical pathway Lectin pathway Alternative pathway

Ag:antibody complex Carbohydrate structures Pathogen surfaces

Complement activation

Recruitment ofinflammatory cells

Opsonization of pathogens Killing of pathogens

Peter Garred

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C9

C5b-8

Microorganism or altered self cell

C3bC4bC2b

C4,C2 C3

Membrane attack complex

MASPs

Lectin pathway attack

C5a

C3b

MASPs

Peter Garred

CL-11 MBL Ficolin-1 Ficolin-2 Ficolin-3

Synonyms Collectin-11

CL-K1

Mannose-binding

lectin

M-Ficolin

P35 related antigen

L-Ficolin

EBP-37

Hucolin

P35

H-Ficolin

Hakata antigen

Thermolabile β2-macroglycoprotein

Gene name COLEC11 MBL2 FCN1 FCN2 FCN3

Gene location 2p25.2 10q11-q21 9q34 9q34 1p36

Tissue expression Ubiquitously Liver Myeloid cells Liver Liver /lung

Binding specificity Fucose

Mannose

GlcNAc

Mannose

GlcNAc

Acetylated groups

Sialic acid

GlcNAc

Acetylated groups

N-acetyllactosamine

Acetylated groups

Plasma conc. (µg/ml) 0.28 1 0.25 5 25

Characteristics of the lectin pathway initiators

Peter Garred

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Models of lectin pathway activation.

Héja D et al. PNAS 2012;109:10498-10503

©2012 by National Academy of Sciences

MASP-1 (MASP1 isoform 1)• activates MASP-2• cleaves C2• cleaves fibrinogen to fibrin, activates fXIII• cleaves high molecular weight kininogen to bradykinin• activates PAR4 on endothelial cellsMASP-3 (MASP1 isoform 2) • cleaves insulin-like growth factor binding protein 5 in vitro• function unknownMAP-1 (MAp44) (MASP1 isoform 3) • inhibits lectin pathway complement activation and thrombogenesis• function unknown

MASP1 gene (chromosome 3q27-q28)

MASP2 gene (chromosome 1p36.3-p36.2)

MASPs: 2 genes, 5 gene products

MASP-2 (MASP2-isoform 1)• cleaves C4 and C2 to generate convertase C4bC2a• cleaves prothrombin to thrombinsMAP (MAp19) or MAP-2 (MASP2 isoform 2)• function unknown Peter Garred

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3MC syndrome=Mingarelli, Malpuech, Michels and Carnevale syndromes

Characteristic facial and cranial dysmorphism, cleft lip and/or palate, learning

disability and genital, limb and muscle abnormalities and many more

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What about ficolin-3?

History of Ficolin-3

1973 Epstein and Tan and 1978 Inaba and Okuchi

Discovers the HAKATA ANTIGEN present in all human sera, (more than 500.000 Japanese and 40.000 Swedesinvestigated), which reacts with an autoantibody present in sera from some SLE patients

The HAKATA ANTIGEN disappears during SLEdisease flares associated with complement activation and is normalized during remission

Peter Garred

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0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

0 2 4 6 8 102 4 6 8 10

Ficolin/MBL (µg/ml)

C4 deposition(OD490n

m)

Ficolin-1Ficolin-1

Ficolin-2Ficolin-2

Ficolin-3Ficolin-3

MBLMBL

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Activation of the lectin complement pathway

Hummelshøj et al. 2008

0

10

20

30

40

50

Fico

lin-3

µg/

ml

Ficolin-3 serum concentration in healthy donors

Munthe-Fog et al. 2008

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Ficolin-3 multimer

FCN3 WT

FCN3 L117fs

Variation of FCN3 (FCN3+1637delC - FCN3 L117fs)

Hummelshøj, Munthe-Fog et al. 2005Allele frequency 0.01, homozygosity expected in 1:10,000

NEJM, 360:2637-2644, 2009

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Search for FCN3 deletion variant in patients with suspected immunodeficiencies

• A total of 1282 patients referred to Department of Clinical Immunology over a period of 12 years for routine immunologic investigation of various immunodeficiencies (not HIV related)

• Sequencing of exon 5 of FCN3 in all 1282 patients

Munthe-Fog, Hummelshøj, Honoré et al., 2009, NEJM

Genotyping of 1282 patients with suspected primary immunodeficiencies

C/C

n=115

C/-

n=23

-/-

n=1

0

10

20

30

40

Ficolin

-3 µg/m

l

P < 0.001

Allele frequency of minor allele = 0.01 � 1 homozygous patient

Munthe-Fog, Hummelshøj, Honoré et al. NEJM, 2009

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Serum Ficolin-3 of index patient and the family

Polyclonal anti-Ficolin-3 Ab

Lanes

1: Heterozygous sister

2: Wild-type sister

3: Heterozygous mother

4: Heterozygous father

5: Index patient

6: Wild-type control

7: Wild-type rFicolin-3

kDa

25

37

50

75

Red

uced

S S M F Pt C Rec

250

150

100

75

5037

25

Unreduced

kDa S S M F Pt C Rec

Munthe-Fog, Hummelshøj, Honoré et al., NEJM, 2009

Medical history of index patient

• 32-years old man (unrelated parents of Macedonian/Albanian origin)

• Since early childhood– Repeated lower respiratory tract infections

• Since age 17– Recurring warts on his fingers

• Age 20– Spleen removed because of unexplained thrombocytopenia

• Age 26– Treated for bilateral frontal cerebral abscesses with non-hemolytic

streptococci• Since age 26

– Several episodes of bacterial pneumonia requiring hospital admission– Severe bronchiectasis and pulmonary fibrosis– Progressively decreased lung capacity and obstructive lung disease

Munthe-Fog, Hummelshøj, Honoré et al., NEJM 2009

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Possible consequences of Ficolin-3 deficiency

Phagocyte

Ficolin

receptor?

Dying cell

Normal cell

complement activation

via associated serine

proteases (MASPs) Clearance of

Cellular debris

Phagocytosis

Peter Garred

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Ficolin-3 specific complement activation ELISA

MASPs

Complement activation

AcBSA

Ficolin-3 binding to acetylated compounds

Hein et al. 2010, PLoS ONE

Ficolin-3 specific complement activation ELISA

Hein et al. 2010, PLoS ONE

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Lectin pathway

� Five recognition molecules exist

MBL, CL-11, ficolin-1, ficolin-2 and ficolin-3

� Two genes encodes 5 MASP gene products

MASP-1, MASP-2, MASP-3, MAP-1 and sMAP

� Lectin pathway activates complement, coagulation and kallikrein systems

� Ficolin-3 is the most abundant and most powerful molecule in the human lectin pathway

Peter Garred