Plasma cell dyscrasia and renal amyloidosis:
What we can learn from basic science
Per Westermark
Rudbeck Laboratory
Uppsala University, Sweden
Definition of Amyloid
• Characteristic ultrastructure
• Cross b-Sheet Fibrils (X-ray diffraction)
• Binding of Congo red with resulting green
birefringence
• Usually extracellular deposits
• In vivo
Folding intermediate
Toxic oligomer
Newly synthesized
Folded, functional protein
protein
Normal function and metabolism
Amyloid fibrils
Merlini & Westermark, JIM 2004
Importance of seeding
Steady state
SEED
Elongation
Lag phase
TIME
The word ’amyloid’ is used widely:
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•
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Amyloid in deposits (”real” amyloid)
Synthetic ”amyloid”
Amyloid-like material in nature (silk etc)
”Functional amyloid”
Amyloid fibril proteins and their precursors in human
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AL
Immunoglobulin light chain
Systemic
AH
Immunoglobulin heavy chain
Systemic
Aβ2M
β2-microglobulin
Systemic
ATTR
Transthyretin
Systemic
AA
(Apo)serum AA
Systemic
AApoAI
Apolipoprotein AI
Systemic
AApoAII Apolipoprotein AII
Systemic
AApoAIV Apolipoprotein AIV
Systemic
AGel
Gelsolin
Systemic
ALys
Lysozyme
Systemic
AFib
Fibrinogen α-chain
Systemic
ACys
Cystatin C
Systemic
ABri
ABriPP
Systemic
ADan*
ADanPP
Systemic
ALect2
Leucocyte chemotactic factor 2
Systemic
Aβ
Aβ protein precursor (AβPP)
Brain
APrP
Prion protein
Brain
ACal
(Pro)calcitonin
C-cell thyroid tumors
AIAPP
Islet amyloid polypeptide (amylin)
Islets of Langerhans, insulinomas
AANF
Atrial natriuretic factor
Cardiac atria
APro
Prolactin
Aging pituitary, prolactinomas
AIns
Insulin
Skin (Iatrogenic)
AMed
Lactadherin
Aortic media
AKer
Kerato-epithelin
Cornea, familial
ALac
Lactoferrin
Cornea
AOaap
Odontogenic ameloblast-ass. protein
Odontogenic tumors
ASemI
Semenogelin I
Vesicula seminalis
Validation of the 3D profile method for prediction of fibrillizing segments.
Goldschmidt L et al. PNAS 2010;107:3487-3492
©2010 by National Academy of Sciences
Some protein factors important for
amyloid fibril formation
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b-sheet propensity
Hydrophobicity
Charge
Destabilisation, e.g. by mutation
Concentration
Degradation
Systemic amyloidosis
• 15 different proteins known
• Precursor circulating in plasma
• The precursor may be modified before
deposition, e.g. by truncation
• Depositions in many organs
Human systemic amyloid fibril proteins and their precursors
AL
AH
Ab2M
ATTR
AA
AApoAI
AApoAII
AApoAIV
AGel
ALys
AFib
ACys
ABri
ADan
ALECT2
Immunoglobulin light chain
Immunoglobulin heavy chain
b2-microglobulin
Transthyretin
Serum AA
Apolipoprotein AI
Apolipoprotein AII
Apolipoprotein AIV
Gelsolin
Lysozyme
Fibrinogen a-chain
Cystatin C
ABri PP
ADan PP
Lecocyte chemotactic factor 2
Primary; myeloma associated
Primary; myeloma associated
Hemodialysis
Familial; senile systemic
Secondary, reactive
Familial
Familial
Sporadic?
Familial
Familial
Familial
Familial
Familial
Familial
Sporadic
Other components in amyloid:
• Amyloid P-component:
AP = SAP is a glycoprotein, produced by the liver. Up to 10% of
the dry substance can consist of SAP
• Proteoglycans:
Heparan sulfate proteoglycan (HSPG)
• Apolipoprotein E
• Other components
Systemic AL amyloidosis
• Extreme variation in deposition pattern
• Sometimes enormous amounts of amyloid
(kilograms)
• Life threatening
• Early diagnosis important
Immunoglobulin molecule
Antibody molecule
Light chain structure
Fab: antigen binding
Variable (VL)
heavy chain , H
light chain , L
VH
variable region, V
VL
CH
constant region, C
CL
CH
Fc: mediates the effect
FR
CDR
Constant (CL)
AL amyloidosis = primary and myeloma
associated amyloidosis
• Primarely a plasma cell disorder: monoclonal
expansion
• Monoclonal immunoglobulin light chain
• Manifests with extreme variations: heart, liver,
kidneys, peripheral nerves etc.
• Treatment with melphalane + prednisolone
and/or peripheral stem cell transplantation
AL amyloid
Each protein is unique; no two sequences are identical:
About 50 different genes for light chains
Somatic mutations
Therefore it is not surprising that each patient has a
unique disease
Properties of the amyloid vary from patient to patient
Affinity for Congo red varies from very weak to strong.
The same is true for the green birefringence in
polarized light
Germ line
Germ line
Enqvist et al., PLoS ONE 2007
Immunoglobulin light chain and AL protein
Variable segment
Constant segment
N
C
Major part of amyloid fibril AL protein is encircled
Fragmentation pattern of monoclonal light chains in
six different AL proteins of kappa type
Enqvist et al., J. Path. 219:473; 2009
Distribution of amyloid in patients with AL-amyloidosis
coming from the two genes O18-O8 and L2-L16
Enqvist et al.,
PLoS ONE 2007
Examples of questions to be answered
• What determines fibril formation from an
immunoglobulin light chain?
• Is cleavage of the light chain of importance?
• What initiates fibril formation? Nidus?
• What determines the deposition pattern?
• Are there toxic oligomers in AL amyloidosis
• Which is the role of HSPG in AL amyloidosis?
Amyloid Diagnostics
Biopsy
Rectum
Subcutis (abdomen)
surgical
needle (not too thin)
Organ with symptom: kidney, liver, skin
Source of errors:
Under or over diagnostics
Insufficient material
Incorrect staining
Errors in microscope (light, polarizers)
Unexperienced examiner
Determination of amyloid type
• Direct method necessary
N Engl J Med 2002, 346:1786-1791.
Misdiagnosis of hereditary amyloidosis as AL (primary) amyloidosis
Lachmann HJ, Booth DR, Booth SE, Bybee A, Gilbertson JA, Gillmore
JD, Pepys MB, Hawkins
Out of 350 patienter with
preliminary diagnosis of AL
amyloidosis 34 (9.7%) had familial
forms!
18 AFib
2 AApoAI
13 ATTR
1 ALys
Typing of amyloid
• Clinical findings: Not enough!
• Immunohistochemistry: Sometimes good but there
are pitfalls
• Immune electron microscopy
• Immunochemistry: Western blot, ELISA
• Mass spectrometry and/or N-terminal sequence
analysis after protein extraction
Surgical Fat Tissue Biopsy For Typing and
for Protein Studies
Immunohistochemistry
Limitations with commercial antibodies
Light chain antibodies: directed against the Cterminal part
Own antibodies: Mab Pwlam
AA
ATTR
AA-amyloid, MAB Sne5
AL-amyloid lambda, MAB PWlam
AL-amyloid lambda, MAB PWlam
AL-amyloid lambda, MAB PWlam
ATTR-amyloid, polyklonal 1898
Mass spectrometry
Identification of specific peptides,
e.g. after tryptic digestion
Laser Dissection Microscopy
Enqvist, Hellman et al., unpublished
Amyloid fibril proteins and their precursors in human
_______________________________________________________________________________________
AL
Immunoglobulin light chain
Systemic
AH
Immunoglobulin heavy chain
Systemic
Aβ2M
β2-microglobulin
Systemic
ATTR
Transthyretin
Systemic
AA
(Apo)serum AA
Systemic
AApoAI
Apolipoprotein AI
Systemic
AApoAII Apolipoprotein AII
Systemic
AApoAIV Apolipoprotein AIV
Systemic
AGel
Gelsolin
Systemic
ALys
Lysozyme
Systemic
AFib
Fibrinogen α-chain
Systemic
ACys
Cystatin C
Systemic
ABri
ABriPP
Systemic
ADan*
ADanPP
Systemic
ALect2
Leucocyte chemotactic factor 2
Systemic
Aβ
Aβ protein precursor (AβPP)
Brain
APrP
Prion protein
Brain
ACal
(Pro)calcitonin
C-cell thyroid tumors
AIAPP
Islet amyloid polypeptide (amylin)
Islets of Langerhans, insulinomas
AANF
Atrial natriuretic factor
Cardiac atria
APro
Prolactin
Aging pituitary, prolactinomas
AIns
Insulin
Skin (Iatrogenic)
AMed
Lactadherin
Aortic media
AKer
Kerato-epithelin
Cornea, familial
ALac
Lactoferrin
Cornea
AOaap
Odontogenic ameloblast-ass. protein
Odontogenic tumors
ASemI
Semenogelin I
Vesicula seminalis
Future Directions
Other amyloid markers (e.g.
polyelectrolytes)
Proteomics
Molecule-specific diagnosis?
Individe-specific treatment?
Thanks to
Knut Sletten, Oslo, Norway
Ulf Hellman, Uppsala
Charles Murphy, Knoxville, TN
Gunilla T. Westermark, Uppsala
Stina Enqvist, Uppsala
All former and present graduate students
All other coworkers
Informa Healthcare
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