BIochtmlca et Blophystca Acta, 1026 (1990) 113-116

113

Elsevaer BBAMEM 74917

Membrane specific carbonic anhydrase (CAIV) expression in human tissues N.D

C a r t e r 1, A F r y e r

2, A . G .

Grant

3, R . H u m e

4, R . G .

S t r a n g e 2 a n d P.J. W l s t r a n d

5

1 Department of Chdd Health, St George's Hospttal Medical School, London (U K), 2 Chmcal Btochemtstry Research Laboratory School of Postgraduate Medicine, Untoerslty of Keele, Staffs (U K ), 3 Department of Surgery, St George's Hospttal Medical School, London (U K ), 4 Department of ChlM L:fe and Health, Umverslty of Edinburgh, Edinburgh (U K) and 5 Institute of Medtcal Pharmacology, Uppsala (Sweden)

(Received21 December1989)

Key words Carbomc anhydrase, Enzymeexpression, (Human) Membrane-bound carbonic anhydrase IV (CAIV) expression has been evaluated in a range of fetal and adult human tissues and in cell culture. All tissues tested showed expression of CAIV, assessed by Western blotting, with a single immunodetected band at 55 kDa. The levels varied in fetal lung and liver during development and in various zones of the fetal brain. CAIV was dearly expressed in lung, pancreatic tumour and skin cell cultures.

Introduction Carbonic anhydrase (EC 4 2 1 1), an efficient catalyst of the reaction, H 2 0 + CO 2 ~ H + + H C O 3 , is present at high levels m erythrocytes and electrolyte-transporting epitheha where it is beheved to mediate the transfer of CO 2, H ÷, HCO~- and C1- [1] A variety of loci encode the isoenzymes identified m mammahan tissues These include CAII which is ubiquitously expressed in mammalian ceils and CAI and CAIII where expression is largely restncted to erythrocytes and skeletal muscle, respecUvely [2] More recently, mltochondnal (CAV) and sahvary (CAVI) lsoforms have been identified as well as the membrane-associated enzyme, CAIV, which is the subject of this commumcatlon [2] In man, CAIV has been identified m the rmcrowlh and basal mfoldmgs of renal tubular cells and this isoform has recently been purified and charactensed [3] An apparently homologous enzyme [4] has also been purified from bovme and adult human lung It is proposed that the CAIV isoenzyme Is p n m a n l y responsible for the reabsorptxon of HCO 3- in the proximal tubule and is Involved In the formation of lung hqmd during fetal hfe We now report, for the first time, the demonstrauon of CAIV expression in a wide range of fetal, adult,

Correspondence N D Carter, Department of Chtld Health, St George's Hospital Medical School, Cranmer Terrace, London, SW17 ORE, UK

normal and mahgnant human cells The ubiquitous expression of the lsoenzyme has enabled us to make new proposals regarding it's function Material and Methods Source o f spectmens

Samples of lung, liver, kidney and brain were obtained w~thln 4 h of death from aborted fetuses (10-22 weeks gestation) following terrmnatlon of pregnancy, premature and term infants (24-42 weeks gestataon) who died in the neonatal pertod and infants who suffered sudden infant death syndrome Red blood cells were also taken from fetuses, neonates and infants Blood specimens were obtained from abortuses (10-22 weeks gestation) by cardiac puncture within 2 h of delivery and from neonates (24-42 weeks gestation) by vempuncture within 24 h of birth as part of an infection screen m infants without overt neurological problems Blood samples were centrifuged at 3000 rpm at room temperature for 5 mmn and the supernatant plasma and buffy coat removed The red cells were resuspended in 154 mM NaC1 and recentnfuged The supernatant was discarded and this wash procedure repeated twine more The pellet was then stored at - 70 o C until required A careful estimate of developmental age was made m each fetus, based on size (mcludmg crown-heel, crownrump and heel-toe measurements), menstrual history and ultrasound In neonates, gestational age was assessed by the Dubowltz score

0005-2736/90/$03 50 © 1990 ElsexaerScmncePubhshers B V (BlomedmalDlvaslon)

114 Approval to take samples was obtmned from the Reproductive Medicine Ethics Committee of the Simpson Memorial Maternity Pavilion, Royal Infirmary, Edinburgh Samples from adult pancreas, colon, kidney were obtained at post mortem or surgical biopsy Samples are stored at - 70 ° C before use

TABLE I

Summary of relatwe C A I V expresston m adult and fetal tzssues and m t~ssue culture +/signs represent relatwe level compared with a standard lodney me mbra ne preparatmn 1 A dul t

2 Tissue culture

Preparation of ttssue homogenates Tissues, stored at - 7 0 ° C , were allowed to thaw shghtly and a sample was removed and washed in ice-cold homogemsatlon buffer to remove superficial blood contamination and then chopped into small pieces using scissors Sample size vaned (0 25 to 2 g) with the tissue and for fetal samples, gestatlonal age Tns-HC1 buffer (20 mM, p H 7 20) contaimng sucrose (250 raM), E D T A (0 1 mM) and reduced glutathlone (1 mM) were added to the tissue pieces which were homogenised on ice and centrifuged (2000 × g 4 ° C, 10 mm) The supernatant provided the membrane fraction for analysis by Western blotting Lung and pancreattc carcmoma cell culture Lung tissue was obtained under sterile conditions from 15 fetuses between 12 and 18 weeks gestation Cubes of lung tissue of 1 - 2 m m were cultured at the a l r / m e d m m interface on lens paper supported by steel gnds in organ culture dishes The cultures were maintamed for 2 - 1 4 days in serum-free Glasgow Modified Eagle Medium (MEM) or m M E M supplemented with 20% fetal calf serum Cultures were maintained at 37 o C in 5% CO2/95% mr and media were changed daily All media contained pemclllln and streptomycin (both 100 I U/ml) Pancreatic tumour cells were grown and harvested as previously described [5] Cultured skin fibroblasts were kindly supplied by the S W Thames Regional Cytogenetics U m t Productton of CAIV antiserum CAIV (molecular mass 35 kDa) punfled as described [3] was used to raise a rabbit polyclonal antiserum Ttus antiserum fmled to detect any band at 28 k D a (the molecular mass of CAII) on Western blots However, restalnmg of immunoblots with anti C A I I serum gave a striking band at 28 k D a This experiment therefore excludes cross reaction of CAIV antibodies with C A I I isoenzyme under the conditions used Immunoblottmg SDS-dlscontinuous polyacrylamlde gel electrophoresis was performed in 10% gels Before loading, the protein content of samples was equahsed by appropriate dilution wath 154 m M NaC1 Proteins were

3 Fetal ussues

Colon (normal and mahgnant) + + + Pancreas (normal and malignant) + + + Kadney membrane vesicles + + + Fetal pneumatocytes + + + Slon fibroblasts + + + Ger pancreatic tumour hne + + + (a) Liver 15 weeks + 40 weeks + + + 40 + 9 weeks + + + + (b) Lung 1 5 weeks + + + + 28 weeks + 40 weeks +/ 4 0 + 1 5 weeks +/(c) Kadney 15 weeks + + + 28 weeks + + + 40 weeks + + + 40 + 84 weeks + + + (d) Brain (single 19 week fetus) Cerebellum + + + Lateral chorold plexus + Cerebrum Lateral inner + / and outer wall +/ Medial than cerebral w a l l + / + Germinal emmanence +/ Thalamus +

blotted onto Nitrocellulose membranes (Hybond C, A m e r s h a m International) using the Novablot system (Pharmacla L K B Biotechnology) CAIV was visuahsed using an alkahne phosphatase system

Results C A I V was detected by lmmunoblottlng in a number of fetal, normal and mahgnant adult h u m a n tissues and in cultured cells The enzyme was Vlsuahsed as a single protein band with a m o n o m e r molecular mass of 55 k D a The 'overall' comparative dlstnbutlon is summarlsed in Table I Since samples were first equahsed for protein content, the intensity of bands dunng development can be compared In fetal hfe CAIV was detected in all tissues tested except red cells Levels of lung CAIV were observed to decrease through fetal hfe, whereas levels m hver increased (Fig 1) C A I V levels m fetal kidney appeared to r e m a m constant through gestation In brain samples obtained from a single individual (19 weeks gestation), expression was strongest in cerebellum C A I V was clearly expressed in cultured skin, pneumatocytes and Ger tumour cells (Fig 1) Fig 1 also shows CAIV expression m normal and mahgnant pan-

115 CARBONICANHYDRASEIV IN HUMANTISSUE Fetal Brain 1

2

3

4

5

6

7

8

Fetal

Liver 12

14

16

24

27

+8

+13

+19

std

weeks gestatmn

Fetal Lung

~-~

weeks

22

24

16

28

24

32

27

34

34

40

gestataon

•~

Fetal Kidney

40

gestation

weeks

Adult Tissues

CAIV mwt 55,000

1

2

3

4

5

6

7

Fig 1 Western blots of whole homogenates from human tissue sand cell extracts Protein concentrations have been normahsed to enable comparison of relattve CAIV concentratton 'Adult Ussuelanes' were 1, kadney membrane vesicles (standard), 2, colon carcmoma, 3, pancreauc carcinoma, 4, pancreatic carcinoma, 5, normal pancreas, 6, normal colon, 7, Ger pancreatic tumour line 'Fetal brmn lanes' 1, kadney membrane vesicles (standard), 2, thalamus, 3, gerrmnal emmlnence, 4, medml thin cerebral wall, 5, cerebrum (lateral outer wall), 6, cerebrum (lateral tuner wall), 7, lateral chormd plexus, 8, cerebellum

creas and colon, showing equal expression in these tissues Discussion

Carbomc anhydrase IV was detected, by Western blotting, in the particulate fraction of all tissues examaned, except red blood cells In each case a band with a monomer molecular mass of about 55 k D a was detected Other workers have found the bovine enzyme to have a smular molecular mass However, esttmates of molecular mass based on amano acid composition suggest that a 20 kDa component represents carbohydrate content [4] The consistency of molecular weight values of human CAIV in diverse tissues would suggest that the carbohydrate (or other hgand) content is constant

but tins wdl need to be confirmed by sequence analysis If tins extra 20 k D a component (added to the native 35 kDa enzyme) is involved in anchoring the CAIV isozyme to the cell membrane, the fact that it is slmdar in all tissues tested indicates a common structure for the enzyme-membrane complex It has been previously suggested that CA may contribute to lung hquld formation in fetal hfe [6] and the precipitate decrease in expression of CAIV at 28 weeks (Fig 1) suggests a possible role for the enzyme in lung during the first two trimesters, but tins does not necessarily point to a role In lung liquid formation Fetal pneumatocytes expressed consistent levels of CAIV through 9 days m culture with only manor fluctuations The enzyme appeared to be more strongly expressed if fetal calf serum was present in the culture medium, but tins will need to be confirmed by more quantitative assay Slon fibroblasts expressed constant levels of CAIV d u n n g culture Only manor differences of CAIV expression in other fetal tissues was observed, for example fetal hver showed a small but consistent increase in level through development Kidney showed more or less consistent expression through fetal life Different areas of the fetal brain vaned in CAIV expression (Fig 1) CAIV bands in immunoblots of fetal hver were consistently 'fuzzy' (Fig 1) and we speculate that tins is because of residual binding of the enzyme to membrane fragments dunng electrophoresls Blotting of purified kidney membrane vesicles gave clearer bands (Fig 1) and restmning of blots w~th antiserum to cytosohc CAII produced very sharp zones of lmmunoreactlon at 28 kDa Tins result suggests that CAIV membrane associating properties may indeed cause the lack of lmmunoblot defmation The true function of carbomc anhydrases I - I I I has been the subject of much conjecture (for review, see Ref 2) and the most hkely (major) role of CAIV would seem to be as a membrane-bound enzyme controlhng H ÷ and H C O 3- transport Tltls has been assumed to be the function of CAIV in lung and lodney epithehal membranes [3] CAIV has been found in both membrane and endoplasmlc retlculum fractions of the kadney in man and In the rat, both brush border and basolateral membranes of tubular cells contaan a CAIV like enzyme However, the unexpectedly ubiquitous distribution of this enzyme found in the present study as well as previous data showing particulate carbomc anhydrase activity in bram, muscle and hver (winch seems hkely to represent at least m part CAIV [7,8]) may indicate a 'housekeeping role' m the cell, possibly in the endoplasmac retlculum It has been proposed that CAIV can function as an 1on channel [9] and the associated membrane bound CO 2 hydrase activity in muscle, for example, related to calcium flux in the sarcoplasmac retlculum [10] may provide a umque role for the enzyme m channeling calcium into the sarco-

116 p l a s n u c retlculum d u r i n g the phases of muscle c o n t r a c tion Thus C A I V m a y fulfil different roles in different tissues T h e h k e l y avaalabthty of c D N A clones for C A I V in the near future, c o u p l e d with expression an tissue culture wall m a k e a useful system for s t u d y i n g c o n t r o l a n d also d e f i m n g the funcUon of C A I V in r e l a t i o n to the o t h e r soluble c a r b o m c a n h y d r a s e s in the s a m e cell

Acknowledgements W e gratefully a c k n o w l e d g e g r a n t s u p p o r t as follows N a t i o n a l K i d n e y R e s e a r c h F u n d (N D C ) , Swedish M e d i c a l R e s e a r c h C o u n c i l ( G r a n t N o 2874, P J W ), British L u n g F o u n d a t i o n ( R S ) a n d Chest, H e a r t a n d Stroke A s s o c i a t i o n ( R S )

References 1 2 3 4 5 6 7 8

9 10

Carter. N and Jeffery. S (1985) Blochem Soc Trans 13. 531-533 Tasluan. R (1989) BloEssays 10. 186-192 Wlstrand. P J and Knuttlla. K -G (1989) I~dney Int 35. 851-859 Wtutney. P L and Bnggle. TV (1982)J Blol Chem 257. 1205612059 Grant. A G. Duke. D and Hermon-Taylor. J (1979) Br J Cancer 39. 143-151 Lonnerholm. G and Wlstrand. P (1982) Pedmtr Res 16. 407-411 Saplrstem. V S. Strocctu. P and G-albert. J M (1984) Am N Y Acad So 429. 481-493 Garcla-Mann. J J. Perez-Barnocanal. F. Garcla. A. Serrano. M A . Reguelro. P and Esteller. A (1988) Btoctum Blophys Acta 945. 17-22 Dlaz. E. Sandblom. J P and Wlstrand. P J (1982) Acta Physlol Scand 116. 461-463 Gros. G and Dodgson. S (1988)Annu Rev Physlol 50. 669-694