MBL Pathway

1
MANNOSE-BINDING LECTIN PATHWAY
Gary C. Pien, MD/PhD Division of
Allergy/Immunology Childrens Hospital of
Philadelphia 34th St and Civic Center
Blvd Philadelphia, PA 19104
2
COMPLEMENT ACTIVATION PATHWAYS
Janeway, C, et al, Immunobiology, New York
Garland Science, 2005.
3
MANNOSE-BINDING LECTIN (MBL)

• pattern recognition receptor
• recognizes pathogen-associated molecular patterns
  (PAMPs)
• member of collectin sub-family of C-type lectins,
  such as surfactants A/D
• serum protein
• synthesized primarily in liver
• acute phase reactant
• rises up to 3x in 1-2 weeks
• multimers of homotrimers
• collagen-like helix domain (stalk)
• ?-helical coiled coil (neck)
• carbohydrate-recognition domain (CRD)
• structurally homologous to C1q

Janeway, C, et al, Immunobiology, New York
Garland Science, 2005.
4
MBL STRUCTURE AND FUNCTION

• binding sites recognize D-mannose, L-fucose,
  N-acetylglucosamine
• binding sites have fixed orientation and spacing
  (45 Å)
• ligands must have correct orientation and spacing
• provides antigen specificity
• structures found only on microbes and not on
  host cells overstated?

Janeway, C, et al, Immunobiology, New York
Garland Science, 2005.
5
MBL LIGANDS
Worthley, DL, et al, 2006, World J Gastroenterol
126420 Takashi, K, et al, 2006, Curr Opin
Immunol 1816.
6
MBL OPSONIZATION

• MBL pathway homologous to classical complement
  pathway
• acts as an ante-antibody
• MBL binds target carbohydrates on microbial
  surfaces
• recruits MBL-associated serine proteases (MASPs)
  to site
• MBLMASP complex activates complement, leading to
  downstream anti-microbial functions

Takashi, K, et al, 2006, Curr Opin Immunol 1816.
7
MASPs 2 GENES, 4 PRODUCTS

• MASP1
• can cleave C2, somewhat C3
• function unknown
• MASP3
• alternative splice product of MASP1 gene
• lacks serine protease domain
• function unknown
• MASP2
• minimal functional unit MASP2 homodimer two
  MBL homotrimers
• cleaves C4 and C2 to generate C3 convertase
  (C4bC2b)
• MAp19
• alternative splice product of MASP2 gene
• function unknown

Sorensen R, et al, 2005, Springer Semin
Immunopathol 27299.
8
MBLMASP TULIPS

• MASPs are predominantly synthesized in liver
• form Ca2-dependent homodimers
• unknown why heterodimers do not occur
• MASP1 and MAp19 associate with low oligomers of
  MBL
• MASP3 and MASP2 associate with higher order
  oligomers
• MASPs can also associate with ficolins (another
  group of PAMP binders)
• binding of MBLMASP complex to ligand activates
  serine protease
• unclear mechanism for how this occurs

Sorensen R, et al, 2005, Springer Semin
Immunopathol 27299 Janeway, C, et al,
Immunobiology, New York Garland Science, 2005.
9
COMPLEMENT ACTIVATION

• activated MBLMASP cleaves C4, C2 to form C3
  convertase
• C3 convertase is bound to surface of pathogen
• cleaves other factors to produce effector
  functions

Janeway, C, et al, Immunobiology, New York
Garland Science, 2005.
10
MEMBRANE-ATTACK COMPLEX C5b, C6, C7, C8, C9
Janeway, C, et al, Immunobiology, New York
Garland Science, 2005.
11
AGE AND MBL LEVELS

• at birth, MBL level is 2/3 of adult concentration
• adult level reached at 1 month of age
• small decline in levels through adulthood

Ip WK, et al, 2004, Scand J Immunol 59310.
12
GENETIC VARIATION

• MBL encoded by MBL2 gene
• MBL1 pseudogene
• both closely positioned on chromosome 10
• exons 1-2 encode Gly-Xaa-Yaa triple collagen
  helix motif to form stalk
• exon 3 encodes coiled-coil neck
• exon 4 encodes CRD domain
• polymorphisms exist in exon 1 that affect MBL
  serum levels
• promoter polymorphisms also exist that affect
  expression levels

Garred, P, et al, 2006, Genes and Immunity 785.
13
GENETIC VARIATION

• A is normal allele ? A/A is normal haplotype
• B, C, and D are variant alleles
• O indicates any of the variant alleles
• serum MBL levels correlate with haplotype
• however, people with same genotype may differ in
  MBL levels by 10x

Garred, P, et al, 2006, Genes and Immunity 785
Worthley, DL, et al, 2006, World J Gastroenterol
126420
14
GENETIC VARIATION

• promoter polymorphisms at 3 distinct locations
  H/L, X/Y, P/Q
• 7 possible MBL haplotypes ? 28 possible genotypes
  (diploid)

• HYPA
• LYPA
• LYQA
• LXPA
• HYPD
• LYPB
• LYQC

Garred, P, et al, 2006, Genes and Immunity 785
Thiel, S, et al, 2006, Mol Immunol
4386Kilpatrick, DC, Biochimica 2002, 1572401.
15
GENETIC VARIATION

• promoter polymorphisms at 3 distinct locations
  H/L, X/Y, P/Q
• 7 possible MBL haplotypes ? 28 possible genotypes
  (diploid)

Dommett, RM, et al, 2006, Tissue Antigens 68193.
16
GENETIC VARIATION

• variant MBL alleles have alterations in collagen
  helix stalk
• impaired multimerization
• forms low-order oligomers
• oligomers have preference for MASP1 and MAp19
  cascade failure?
• non-functional and unstable

Eisen, DP and Minchinton RM, 2003, Clin Inf Dis
371496.
17
MBL LEVELS CORRELATE WITH ACTIVITY

• MBL concentration affects activity of complex and
  C4b deposition

Worthley, DL, et al, 2006, World J Gastroenterol
126420
18
MBL DEFICIENCY AND DISEASE ASSOCIATIONS

• MBL linked to human disease based on case report
  in 1968
• female patient with recurrent URI and diarrhea in
  childhood
• no apparent immunodeficiency
• neutrophils cultured in patients serum could not
  phagocytose yeast
• low C3 deposition but normal complement function
• common opsonic defect with impaired complement
  deposition
• defect found in 5-8 of health population
• separately, group purified a protein from rabbit
  liver using yeast mannan
• protein could activate complement when bound to
  mannan surfaces
• led to identification of MBL

Garred, P, et al, 2006, Genes and Immunity 785.
19
MBL DEFICIENCY AND DISEASE ASSOCIATIONS

• what is a physiologically low level?
• ranges from 5 10,000 ng/ml in population
• no individual completely deficient in MBL has
  been reported
• 30 of population has level lt 500 ng/ml
• 10-20 have levels lt 100 ng/ml
• 50-100 ng/ml has been labeled severe MBL
  deficiency
• majority of individuals with low MBL are healthy
• difficult to link MBL deficiency with infection
  susceptibility
• infants have recurrent respiratory infections,
  OM, chronic diarrhea
• MBL important when maternally acquired antibodies
  wane and host responses still maturing?
• clinical significance likely varies depending on
  disease context
• disease susceptibility modifier?

Garred, P, et al, 2006, Genes and Immunity 785.
20
MBL DEFICIENCY AND INFECTIONS
Eisen, DP and Minchinton RM, 2003, Clin Inf Dis
371496.
21
MBL DEFICIENCY AND INFECTIONS
Eisen, DP and Minchinton RM, 2003, Clin Inf Dis
371496.
22
MBL DEFICIENCY AND INFECTIONS
Eisen, DP and Minchinton RM, 2003, Clin Inf Dis
371496.
23
MBL DEFICIENCY AND DISEASE ASSOCIATIONS

• chemotherapy patients
• children with ALL and variant MBL had twice as
  many days of FN
• adults who developed bacteremia or pneumonia
  after chemotherapy had lower levels of MBL than
  those who did not
• low MBL associated with increased rates of severe
  infections and FN in adult cancer patients
• allogeneic hematopoietic stem cell transplant
  patients
• invasive infections occurred more frequently in
  patients with variant MBL alleles

Thiel, S, et al, 2006, Mol Immunol 4386.
24
MBL DEFICIENCY AND DISEASE ASSOCIATIONS

• SIRS / sepsis / MOD
• adults who died in ICU from sepsis/MOD had 3x
  lower MBL levels
• adults with variant MBL genotypes had higher risk
  of sepsis

Thiel, S, et al, 2006, Mol Immunol 4386 Klein,
NJ, 2005, Mol Immunol 42919.
25
MBL DEFICIENCY AND DISEASE ASSOCIATIONS

• autoimmunity
• low MBL levels linked to worse prognosis in RA
• low MBL predisposes to SLE
• low MBL strongly associated with more severe
  celiac disease
• vascular disease
• higher frequency of MBL mutations in patients
  with Kawasaki disease
• higher risk of MI in patients with low MBL levels
• obstetrics
• couples with recurrent spontaneous abortion had
  higher frequency of MBL deficiency in both
  partners
• other studies only found association with
  maternal deficiency

Thiel, S, et al, 2006, Mol Immunol 4386.
26
MASP DEFICIENCY

• patient with MASP2 deficiency
• healthy until age 13
• developed UC
• erythema multiforme bullosum
• SLE?
• 3 episodes of severe pneumococcal pneumonia
• progressive lung fibrosis
• hypocomplementemia
• homozygous D105G mutation in MASP2
• other family members healthy
• had low activity levels in MBL pathway
• were heterozygous for MASP2 mutation

Stengaard-Pedersen, K, et al, 2003, NEJM 349554.
27
MASP DEFICIENCY

• 10 of general (Danish) population is
  heterozygous for D105G mutation (492 pts)
• 50 of normal circulating MASP2 levels
  (heterozygotes 200 ng/ml)
• individuals who are homozygous expected at 11000
• have only 10 of normal circulating MASP2 levels
  ( 50-75 ng/ml)
• no Hong Kong Chinese carried D105G mutation (573
  pts)
• did find another deficiency-associated mutation,
  rare frequency

Kilpatrick, DC, Biochimica et Biophysica Acta
2002, 1572401 Sorensen R, et al, 2005, Springer
Semin Immunopathol 27299.
28
COMPLEMENT DEFICIENCIES - KATE
http//www.clinimmsoc.org/teaching/tips/complement
_deficiencies.pdf
29
PATHWAY DEFECTS AND HUMAN DISEASE
Janeway, C, et al, Immunobiology, New York
Garland Science, 2005.
30
MBL PATHWAY TESTING

• IBT Laboratories (www.ibtreflab.com)
• MBL protein
• sandwich ELISA
• uses monoclonal antibody against oligomeric CRD
• define normal gt 100 ng/ml
• MBL pathway function test
• ELISA C4b deposition assay
• functional assay of initial pathway (MBL, MASP)
• ProGenotyper MBL panel
• rtPCR for MBL variant alleles
• ProGenotyper MASP2 polymorphisms
• DNA sequencing (D105G or entire gene?)

31
MBL REPLACEMENT THERAPY

• NatImmune A/S (www.natimmune.com)
• Danish biotech company
• developed rhMBL
• Phase I clinical trial in 2004
• infused rhMBL into healthy subjects
• no significant adverse events
• serum t½ 30 hours
• Enzon Pharmaceuticals (www.enzon.com)
• US biotech company licensed rhMBL from NatImmune
  in 2005
• Phase I/II clinical trials
• rhMBL prophylaxis and treatment
• oncology and liver transplant patients
• reduce risk of infection

32
CONCLUSIONS

• MBL pathway involved in host defense and possibly
  other functions
• MBL deficiency alone not clearly associated with
  infection risk
• deficiency most clearly associated with increased
  infection risk in immunosuppressed/co-existing
  immunodeficient patients
• deficiency may predispose to infections in
  infancy/early childhood
• low MBL may be susceptibility risk factor for
  other diseases
• MASP2 deficiency has been associated with
  recurrent infections
• n 1 (maybe 2)
• MASP2 may have other functions outside of MBL
  pathway (ficolins)
• MASP2 deficiency may have more prominent
  phenotype than MBL deficiency