genetic disorders of carbonic anhydrases ii and iv prof. william … · but did not affect kidney...

Genetic Disorders

of Carbonic Anhydrases II and IV

Prof. William S. Sly

The screen versions of these slides have full details of copyright and acknowledgements 1

1

Genetic Disorders of Carbonic Anhydrases II and IV

William S. Sly, M.D.Chairman, Department of Biochemistry & Molecular Biology

Saint Louis University School of Medicine

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The carbonic anhydrase (CA) reaction

The reaction existed long before enzymes, when only gases and water covered the earth

CO2 + H2O ↔ HCO3¯ + H+

Uncatalyzed reaction pretty fast, but can be accelerated by enzymes up to 5000-fold

3

American family with first inherited CA deficiency syndrome

Ca se 3Ca se 3Ca se 3Ca se 3 Un a ffected Un a ffected Un a ffected Un a ffected s isters isters isters ister

Ca se Ca se Ca se Ca se 2222 Ca se 1 Ca se 1 Ca se 1 Ca se 1 (p ropositus)(p ropositus)(p ropositus)(p ropositus)

Sly and Shah, MMBID, 8th Ed., Chp. 208, pp. 5331-5343, 2001

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Right tibia and fibula at 2 years of age, showing fracture and osteopetrosis

(first feature)


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We suspected it was a new disease; Why?

1.1.1.1. Re cessive, infantile, malignant, lethal formRe cessive, infantile, malignant, lethal formRe cessive, infantile, malignant, lethal formRe cessive, infantile, malignant, lethal formAll died before age 5 from infection or anemia;No marrow space to make RBCs and WBCs

2.2.2.2. Be nign, autosomal dominant formBe nign, autosomal dominant formBe nign, autosomal dominant formBe nign, autosomal dominant formOften asymptomatic;Usually detected incidentally in adults on X-ray for another reason

• Ou r family had:Ou r family had:Ou r family had:Ou r family had:• Three affected sisters with normal parents• All lived well beyond childhood

Then, only two forms of osteopetrosis were known

Th e refore, we must have something newTh e refore, we must have something newTh e refore, we must have something newTh e refore, we must have something new

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Second feature:Renal Tubular Acidosis (RTA)

Pa tientsPa tientsPa tientsPa tients No rmalsNo rmalsNo rmalsNo rmals

L o w blood pHL o w blood pHL o w blood pHL o w blood pH 7 .1 7 .1 7 .1 7 .1 –––– 7.27.27.27.2 7 .47 .47 .47 .4

L o w plasma bicarbonateL o w plasma bicarbonateL o w plasma bicarbonateL o w plasma bicarbonate 1 2 1 2 1 2 1 2 –––– 20202020 2 2 2 2 2 2 2 2 –––– 24242424

Hi gh urine pHHi gh urine pHHi gh urine pHHi gh urine pH 7 .0 7 .0 7 .0 7 .0 –––– 7.57.57.57.5 < 6 .0< 6 .0< 6 .0< 6 .0


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Third feature: cerebral calcification


CT scan of the head at 33 years of age

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• What single enzyme deficiency could produce such defects in:

1) Bone resorption?2) Renal acidification?3) Brain calcification?

• After another patient led me to ponder this question 10 years later, I hypothesized an inherited defect in carbonic anhydrase

The question:

I called Richard Tashian, an expert on CAs

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14 15

1 2 3 4

, : unaffected : affected

Patterns of erythrocyte CA I and CA IIin affected and control individuals

Sly et al., Proc. Natl. Acad. Sci. USA, 80808080: 2752-2756, 1983

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My first paper on carbonic anhydrases

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Is it always true in osteopetrosis with renal tubular acidosis?

• Yes! Every patient with this phenotype worldwide had CA II deficiency

• Every patient so far had a structural mutationin the CA II gene, making prenatal DNA diagnosis possible

• Treating acidosis improved growth and well-being,but did not greatly alter the course of the disease

• Bone marrow transplant corrected osteopetrosisbut did not affect kidney or brain disease

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Sixteen structural gene mutations in CA II-deficient patients


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The proposed roles of CA II and CA IV in bicarbonate reclamation (A)

and of CA II in distal urinary acidification (B)

A.A.A.A. B.B.B.B.


Proximal tubule Distal tubule

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How do we explain cerebral calcification?

CT scan of the head at 33 years of age


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1983: 50-year anniversary symposium

• Why was I there?

• That year, we reported CA II deficiency, the first human diseasedue to a CA deficiency

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How was carbonic anhydrase discovered?

Ca rbonic anhydrase; Its preparation and propertiesCa rbonic anhydrase; Its preparation and propertiesCa rbonic anhydrase; Its preparation and propertiesCa rbonic anhydrase; Its preparation and propertiesMeldrum NU and Roughton FJW

J Physiol 80808080:113-141, 1933

Th e catalysis of the hydration of carbon dioxide Th e catalysis of the hydration of carbon dioxide Th e catalysis of the hydration of carbon dioxide Th e catalysis of the hydration of carbon dioxide a n d dehydration of carbonic acid a n d dehydration of carbonic acid a n d dehydration of carbonic acid a n d dehydration of carbonic acid b y an enzyme isolated from red blood cellsb y an enzyme isolated from red blood cellsb y an enzyme isolated from red blood cellsb y an enzyme isolated from red blood cells

Stadie WC and O’Brien HJ Biol Chem 103103103103:521-529, 1933

CAs eventually found in every living organism, from bacteria to humans

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Purified enzyme was found to contain zinc

Ca rbonic anhydrase, purification and nature of the enzymeCa rbonic anhydrase, purification and nature of the enzymeCa rbonic anhydrase, purification and nature of the enzymeCa rbonic anhydrase, purification and nature of the enzymeKeilin D and Mann T

Biochem J 34343434:1163-1176, 1940

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Medical uses for CA inhibitors:

• Diuretics– HCO3

¯ + H2O loss in kidney

• Glaucoma– ↓Aqueous humor (H2O)

• Brain swelling– ↓CSF production (H2O)

• Epilepsy– Mechanism ???

Inhibitors showed CA’s function in:

• CO2 transport (lung)

• HCO3¯ reabsorption (kidney)

• HCO3¯ secretion (pancreas)

• H+ secretion (stomach)

• Production of aqueous humor (eye) and cerebrospinal fluid (choroid plexus)

1945 – 1960: the middle years

• Sulfonamide inhibitors defined the physiological roles and led to the development of important drugs

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1960 – 1980: the protein era

• Identification and separation of individual isozymes

• Amino acid sequencing (primary structure)

• X-ray crystal structure – CA I, CA II, etc.

• Immunolocalization of individual CAs

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Other CA family members known by 1983:

• Third isozyme; CA III – a.k.a. basic protein of muscle

• Membrane form; CA IV – resistant to SDS,in lung and kidney

• Mitochondrial form; CA V – in liver and kidney

• Salivary form; CA VI

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In the mid-80’s, my question was:

Why did god give us six enzymes Why did god give us six enzymes Why did god give us six enzymes Why did god give us six enzymes for such a simple reaction? for such a simple reaction? for such a simple reaction? for such a simple reaction?

COCOCOCO2222 + H+ H+ H+ H2222 O ↔ HCOO ↔ HCOO ↔ HCOO ↔ HCO3333---- + H+ H+ H+ H++++

• Actually, we’ve since found there are 15 mammalian CAs,and also several CA-related proteins

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Partial answer to why so many:compartmentalization

• Cytoplasmic: CA I, II, III, VII

• Membrane: CA IV, and later, CA IX, XII, XIV, XV

• Secretory: CA VI

• Mitochondrial: CA V (VA and VB)

– Numbers only indicate order of discovery– Each proved to be the product of a different gene

Other reason for many:varied, tissue-specific expression

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The 260-amino acid CA domainis commonto all CAs

Structure-based sequence alignment of cytoplasmic CA II and membrane-associated, extracellular human CA IV, murine CA IV, human CA XII,and murine CA XIV

Whittington et al., Proc. Natl. Acad. Sci. USA, 98989898: 9545-9550, 2001

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N-terminal amino acid sequences of the catalytic human CAs

Lehtonen J. et al., J. Biol. Chem. 279279279279: 2719-2727; 2004

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C-terminal amino acid sequences of the catalytic human CAs

Lehtonen J. et al., J. Biol. Chem. 279279279279: 2719-2727; 2004

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CA-related proteins (CARPs)have other uses

for the 260-amino acid CA domain

• None of them have CA enzymatic activity but are highly conserved between mouse and man

• CA VIII, CA X and CA XI: have unknown functions

• RPTPs: inactive CA domain serves as ligand-binding domain for signal transduction protein

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Back to CA IV…Why was it of special interest?

• In 1983, it was the only membrane CA known• We purified the human CA IV

and found it to be a 35-kDa GPI-anchored membrane protein expressed on:

– Luminal membranes of epithelial cells in the proximal convoluted tubules in kidney, as we expected,

But we also found it on – Plasma face of endothelial cells of:

– Pulmonary microcapillaries– Choriocapillaris of the eye

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CA IV in the lumen of the proximal renal tubules

Rat kidney cortex – CA IV

Brown et al., Proc. Natl. Acad. Sci. USA 87: 87: 87: 87: 7457-7461, 1990

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Immunofluorescent labeling of CA IV in the choriocapillaris

Courtesy of R. Mullens, Univ. of Iowa

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Cartoon of the CA IV-membrane interaction

Stams et al., Proc. Natl. Acad. Sci. USA, 93939393: 13589-13594, 1996

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31Sly and Shah, MMBID, 8th Ed., Chp. 208, pp. 5331-5343, 2001

Back to American family with the CA II deficiency syndrome

Ca se Ca se Ca se Ca se 3333 Un a ffected Un a ffected Un a ffected Un a ffected s isters isters isters ister

Ca se 2Ca se 2Ca se 2Ca se 2 Ca se 1 Ca se 1 Ca se 1 Ca se 1 (p ropositus)(p ropositus)(p ropositus)(p ropositus)


What CA IV does for you in your proximal tubules

A.A.A.A. B.B.B.B.Proximal tubule Distal tubule

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How to prove it?

We proposed he/she should have what nephrologists call:“Pu re proximal renal tubular acidosis” “Pu re proximal renal tubular acidosis” “Pu re proximal renal tubular acidosis” “Pu re proximal renal tubular acidosis”

Isolated bicarbonaturia without any other featuresof the Fanconi Syndrome, i .e., no aminoaciduria or glycosuria

• We found several such families in the literature

• We obtained DNA, but they did not have a CA IV mutation

• Instead, they had a mutation in the Na:bicarbonate co-transporterin the basolateral membrane

Ra ts! Ra ts! Ra ts! Ra ts!

Find a CA IV deficient patient

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On to the mouse!

• We thought that making a mouse knockout model

would tell us how CA IV works

Answer?

• Rats again – no phenotype in the CA IV knockout!

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This was a big surprise!This was a big surprise!This was a big surprise!This was a big surprise!

Question:Why is CA IV non-essential in kidney?

Answer:Another membrane CA, CA XIV,makes it redundant in kidney

Mouse knockout has no kidney phenotype

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CA IV and CA XIV in kidney proximal tubulesM ouse kidney cortex M ouse kidney cortex M ouse kidney cortex M ouse kidney cortex –––– CA XIVCA XIVCA XIVCA XIVR at kidney cortex R at kidney cortex R at kidney cortex R at kidney cortex –––– CA IVCA IVCA IVCA IV

Kaunisto et al., Kidney Int., 6 16 16 16 1 : 2111-2118, 2002

Brown et al., Proc. Natl. Acad. Sci. USA8 78 78 78 7 : 7457-7461, 1990

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Predicting wrong phenotypefor human CA IV deficiency –

not pure proximal RTA

+ CA XIV+ CA XIV+ CA XIV+ CA XIV

A.A.A.A. B.B.B.B.Proximal tubule Distal tubule

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Summary of CA IV and CA XIV findings in kidney:

• CA IV and CA XIV overlap in distribution in kidney; Deletion of either has no phenotype

• Making the double knockout by crossing the two single knockouts gives a sick mouse

• Physiologists were right that a membrane CA in the lumen is essential for bicarbonate reabsorption…Bu t either one will doBu t either one will doBu t either one will doBu t either one will do

39Parkkila et al., BMC Gastroenterol. 2222: 13-19, 2002

Confocal laser scanning images of CA XIV in the mouse liver

← EC (D)

← SLCs (E and F)

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40

Distribution of CA XIV →

Antibody selectivity

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Colocalization of CA XIV and Müller cell marker, glutamine synthetase, in Müller cells in retina

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ΔΔΔΔpHpHpHpH eeee

P er cent P er cent P er cent P er cent am pl ificationam pl ificationam pl ificationam pl ification

AAAA CCCCBBBB

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43

We thought we were finished with CA IV…

Finally!Finally!Finally!Finally!

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But then, out of the blue…

Raj Ramesar asked for help with a South African familywith autosomal dominant retinitis pigmentosa

• Maps to the CA IV locus

• Is associated with a mutation in the signal sequence at the -5 position relative to the signal sequence cleavage site

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What is retinitis pigmentosa (RP)?

• Genetic disease with progressive loss of vision

• Onset at 15 - 30 years of age

• Rod cells at periphery die first, leading to loss of peripheral vision

• Zones of cell death expand to leave only central, tunnel vision

• May progress to total blindness

• Question: if CA IV is a non-essential gene, how could it be related to retinitis pigmentosa?

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What does the retina look like in RP?

• Retinal cells die, starting at periphery

• Cells full of black melanin pigment invade the dead retinal tissue

• The black depositsare the hallmark of RP

Montgomery G, in: Handbook of Ocular Disease Management, Jobson Pub, 2001

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Mutations in rhodopsin and mechanisms of retinal disorders:

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What is the predicted effect of the R14W mutation

on the CA IV protein sequence?

Rebello et al., Proc. Natl. Acad. Sci. USA, 101101101101: 6617-6622, 2004

Answer:No effect on sequence of the mature protein;Possible effect on signal cleavage and folding

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49

Could we find a precedent for a signal sequence mutation causing an autosomal dominant disease?

Yes, here is one example of two I found:

• Familial hypoparathyroidism from single mutant allele can be caused by:Cys → Arg mutation at -8 position from signal cleavage site of preproparathyroid hormone, which impairs signal sequence cleavage

Co n clusion:Co n clusion:Co n clusion:Co n clusion:Mutant allele must lead to death of parathormone-producing cells in parathyroid,despite presence of the normal allele

Liu et al., J. Biol. Chem., 270270270270: 1624-1628, 1995

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Didn’t we say CA IV is absent in the retina?

Hageman et al., Proc. Natl. Acad. Sci. USA, 88888888: 2716-2720, 1991

Anti-CA II Anti-CA IV

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Yes, but it is highly expressed in the choriocapillaris

Courtesy of R. Mullens, Univ. of Iowa

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Thus, as a working hypothesis for RP17:

• The R14W mutation in the signal sequence in CA IV leads to impaired signal cleavage and folding, which induces the unfolded protein response (UPR), “ER stress”, and eventual apoptosisin endothelial cells

• The retinal disease in RP17 results indirectly from death of capillary endothelial cells and subsequent retinal ischemia

We p roposed that:We p roposed that:We p roposed that:We p roposed that:

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What is the UPR?

• Excess unfolded proteins in the ER produce “ER stress,”

a threat to all living cells

• In response, the cell activates

a signal transduction pathway,

inducing the Unfolded Protein Response (UPR)

• The UPR defends the cell from changes

leading to cell death (apoptosis)

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How does the UPR help the cell handle ER stress?

• By increasing levels of chaperone proteinsthat assist folding

• By increasing the rate of degradation of unfolded proteins

• By decreasing overall protein accumulationby ↓ translation

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To test whether expression of the R14W mutant CA in COS cells

induces the UPR…

• We compared cells transfected with plasmids expressing the R14W vs. wild-type CA IV cDNA for:

1) The amount of CA IV enzyme produced

2) The rate of degradation of newly synthesized enzyme (induction of ER AD)

3) The upregulation of the chaperone protein BiP and the activation of PERK

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The R14W mutation reduces the steady-state level of CA IV protein

• Also, the activity of R14W extract was only 70% that of wild-type


2 µ g2 µ g2 µ g2 µ g 2 µ g2 µ g2 µ g2 µ g

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Turnover of R14W and wild-type CA IV protein in the absence and presence of inhibitor MG 132


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Effect of the R14W mutation on induction of the ER proteins,

BiP and PERK, in transfected COS-7 cells


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Can the observed R14W CA IV-induced ER stress overwhelm cell defensesand lead to cell death (apoptosis)?

• Signs that cells have entered the apoptotic pathway:

– Upregulation of CHOP, a proapoptotic protein – Annexin V binding: indicates cells can no longer

maintain asymmetry of membrane lipids (inside-out phospholipid)

– TUNEL staining: shows increased number of DNA ends produced by fragmentation of the DNA by nucleases

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R14W mutant CA IV induces upregulation of CHOP in transfected COS-7 cells

0000 1 01 01 01 0 2 02 02 02 0 WTWTWTWT R1 4 WR1 4 WR1 4 WR1 4 WTu nicamycinTu nicamycinTu nicamycinTu nicamycin

Tunicamycin treatment = 3 h


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R14W mutant CA IV induces Annexin V and TUNEL staining in transfected COS-7 cells


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So what?

Good question!

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Well…If RP17 is a protein folding disease…

• …it might be treatable with chemical chaperones

• What are chemical chaperones?

• Sm all molecules that enhance protein folding:Sm all molecules that enhance protein folding:Sm all molecules that enhance protein folding:Sm all molecules that enhance protein folding:– By binding to a specific site on the protein,

e.g., enzyme inhibitor (specific for only one protein)– By favorably affecting the intracellular environment

in a general, non-specific way,e.g., phenyl-butyric acid, glycerol

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Will the CA inhibitor, acetazolamide, affect the rate of degradation of R14W mutant CA IV protein?

Bonapace et al., Proc. Natl. Acad. Sci. USA, 101101101101: 12300-12305-, 2004

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Will the general chaperone, 4-phenylbutyric acid (PBA),

affect the biosynthes is and stability of R14W mutant CA IV?

Bonapace et al., Proc. Natl. Acad. Sci. USA, 101101101101: 12300-12305-, 2004Time (min)

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Will CA inhibitors reduce UPR gene upregulation?

Bonapace et al., Proc. Natl. Acad. Sci. USA, 101101101101: 12300-12305-, 2004

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Will chemical chaperones protect cells from apoptosis?

Bonapace et al., Proc. Natl. Acad. Sci. USA, 101101101101: 12300-12305, 2004

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Summary

• Apoptosis-inducing signal sequence mutation in CA IV

was identified in family members with RP17

• Chemical chaperones can reduce its apoptotic effects

in transfected COS cells

• These drugs may delay the onset of or prevent blindness

in family members at risk for RP17

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Future studies• Create mouse model of RP17 to test chaperones (Gul Shah)

• Test generality of general chaperone effects on other diseases associated with signal sequence mutations

• Extend studies to mutations involved in other folding diseases

* All under the skillful direction of my colleague,Abdul Waheed

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Immunofluorescence of transgenic retina

Histological layers of mouse retina

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TUNEL staining of retina from control mouse

Control (untreated) DNAase-treated

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TUNEL staining of retina from R14W CA IV transgenic mouse

Lower sensitivity Higher sensitivity

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genetic disorders of carbonic anhydrases ii and iv prof. william … · but did not affect kidney...

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