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January 1975 The Journal o f P E D I A T R I C S

Plasma membrane components of skin fibroblasts from normal individuals and patients with cystic fibrosis Plasma membranes have been isolated without proteolytic modO%atlon from fibroblast lines derivedfrom patients with C~, from heterozygous parents, and from normal children. The cells had been grown in the presence of 3H- or 14C-labeled L-leucine, D-glucosamine, and L-fueose. Membranes were mixed in suitable combinations to allow comparisons to be made between the different cell types. No differences in the plasma membrane composition, as revealed by divergence in 314-or 14C-profiles, could be detected after gel eleetrophoresis. Identieal protein and glycoprotein components were present in approximately similar amounts in all groups of cells.

M . M a n s o o r B a i g , P h . D . , J o h n J . C e t o r e l l i , B . S . , and R . Michael Roberts, D . P h i l . , * G a i n e s v i l l e , Fla.

MOST OF THE PATHOLOGIC CHANGES and clinical symptoms associated with CF are due to abnormal mucous s e c r e t i o n s in the lungs, p a n c r e a s , and o t h e r mucus-producing glands 1, 2 which have led several investigators to propose that the mucus is either abnormal in quality or produced in abnormally high amounts during the course o f the disease. A l t e r n a t i v e l y , these changes may be a consequence of unusual electrolyte balance in the secretory fluid),4 Because the disease is inherited as an autosomal recessive trait, 2attempts have been made to detect abnormalities in cells isolated from CF individuals, 5-14 including fibroblasts derived from skin. Reports have indicated that the CF fibroblast, its products, or its plasma m e m b r a n e 15may be abnormal in some way, and that the disease may be a primary disorder of the exocrine glands. We decided, therefore, to investigate the plasma membrane composition of CF From the Departments o f Biochemistry and Pediatrics, University o f Florida. This work was supported by grants from the Cystic Fibrosis Foundation, Inc., and from the National Institutes o f Health (AM-15023-01). Dr. Roberts received a Career Development A wardfrom the United States Public Health Service. *Reprintaddress:Department of Biochemistry, J. Hillis Miller Health Center, Universityof Florida, Gainesville,Fla. 32610.

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fibroblasts compared to controls to see if there were any a l t e r a t i o n s in the p r o t e i n s or g l y c o p r o t e i n s p r e s e n t . Changes of this kind might conceivably account for differences in the m o v e m e n t of materials across the cell m e m b r a n e or, because of the carbohydrate-rich nature of the cell surface, reflect any over-all changes in the complex saccharide metabolism of such cells=

Abbreviation used CF: cystic fibrosis

MATERIALS AND METHODS Cell cultures and labeling experiments. Skin biopsies from CF children (aged between 3 and 10 yr) were obtained from patients treated at the C F clinic o f the Jacksonville Baptist Memorial Hospital or a d m i t t e d into the J. Hillis Miller Health Center in Gainesville. All of the CF patients had high sodium concentrations in their sweat and had severe pulmonary or pancreatic involvem e n t . Skin b i o p s i e s o f h e t e r o z y g o t e s w e r e o b t a i n e d f r o m p a r e n t s of C F c h i l d r e n . N o r m a l m a t e r i a l was derived from foreskins. Monolayer cultures were established and, when confluent, they were s u b c u l t u r e d into 75 cm 2 Falcon flasks. All experiments were p e r f o r m e d

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Fig. 1. Profiles for L-leucine incorporation along SDS polyacrylamide gels of plasma membrane preparation from N(3H, --Q --O --) and CF(14C, "'9 "'0 ") fibroblasts. The cell lines employed were 55 (CF) in passage 7 and 35 (N) in passage 6. The total amount of radioactivity recovered from the gels was: 14C,21,500 dpm; 3H, 122,900 dpm. Each gel slice was 0.8 mm. The staining pattern with Amido Black of an unfracti0nated gel is also shown. The cells used here were from HZ line 53, but all others examined have shown identical staining of protein bands. Positions of markers for MW determinations ( x 10-3) are shown by arrows9 on cells which had undergone between six and eight one-to-two passages. Cells were grown at 37 ~ in an atmosphere of 95% air and 5% CO 2. They were maintained on 18 ml Eagle's M i n i m u m Essential Medium in Earle's salts reinforced with 16.7% (v/v) heat-inactivated fetal calf serum (Bioquest, Cockeysville, Md.) and containing antibiotic-antimycotic mixture (Grand Island Biological Company, N. Y.). In a single labeling experiment with L-leucine, eight flasks were each inoculated with 3 x 106ceils from a CF line. Four of these flasks were given 3H-labeled precursor (100 /zCi), the remainder ~4C (20 txCi). This was repeated for the contrasting HZ and N lines. Identical e x p e r i m e n t s were r u n with D-glucosamine and Lfucose.

Plasma membranes were harvested from cells when they were just confluent but not overgrown. This usually took 96 hr. R a d i o c h e m i c a l s . D - [ I - 1 4 C ] - g l u c o s a m i n e (53 tzCi//zmote), D-[I- 3H]-glucosamine (2.6 mCi//xmole), Lr[l-3H]-fucose (0.92 mCi//~mole),L-[/-14C]-leucine (50 /zCi/tzmole) were o b t a i n e d from A m e r s h a m - S e a r l e Corp. L-[l-14C]-fucose (45 /xCi/tzmole) and L-[4-5-3H] leucine (54 mCi//zmole) were products of New England Nuclear Corp. and Schwarz-Mann, respectively. Isolation of surface membranes. Plasma membranes were isolated by the method of Barland and Schroeder, 16 and purified by centrifugation (1 hr; 150 gm) in 30% (w/v) sucrose layered above a 45% (w/v) sucrose bed (25 ml) in a centrifuge with a swinging b u c k e t head. The i n t e r p h a s e region was collected,

74

Baig, Cetorelli, andRoberts

The Journal of Pediatrics January 1975

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Fig. 2. Profiles for D-gmcosamine incorporation along SDS polyacrylamide gels of plasma membrane preparations from CF(14C, "'O"O") and HZ(3H, - - 0 - - Q --) fibroblasts. The cell lines employed were CF 55 in passage 7 and HZ 45 in passage 6. The total amount of radioactivity recovered was:Z4C, 1860 dpm; 3H, 18,900 dpm. Each gel slice was 1.6 mm. diluted with 10--3M NaHCO3, and centrifuged (1 hr; 10,000 g). The m e m b r a n e pellet was dissolved in a m i n i m u m amount of 0.04M Tris-glycine buffer pH 8.3 containing 2% (w/v) sodium dodecyl sulfate and boiled for three minutes. Polyaerylamide gel electrophoresis. Portions of contrastintly labeled, solubilized m e m b r a n e fractions were combined and dialyzed against 0.01M sodium phosphate buffer, pH 7.0, containing 0.1% (w/v) SDS and 0.1% (w/v) mercaptoethanol for 3-5 hr. Polyacrylamide gels (7.5%) containing 0.1% (w/v) SDS and mercapt o e t h a n o l w e r e r u n a c c o r d i n g to t h e p r o c e d u r e described by W e b e r and Osborn, 17 and stained with 0.2% (w/v) Amido-black 10B to detect proteins. For determination of radioactivity, unstained gels were fractionated with an autogel divider (Savant Instruments, Hicksville, N. Y.). In experiments with L-leucine, in which the membranes were highly labeled, the gels (8 cm) were d i v i d e d into a p p r o x i m a t e l y 100 fractions. With L-fucose and D-glucosamine, only half this number of fractions were taken. The gel fractions were

placed overnight in 0.5 ml of water before scintillation counting. Determination of radioactivity. The content of 3H a n d 14C was d e t e r m i n e d after a d d i t i o n o f 5 ml of Toluene-Triton X-100 scintillant ~8 with the use of a Packard Tricarb Scintillation Spectrometer. All results are presented as percentages of the total 14C or 3H recovered from the gels (usually 80-85%). RESULTS SDS polyacrylamide gel electrophoresis was used to separate the component proteins and glycoproteins of the solubilized plasma m e m b r a n e preparations from N, HZ, and CF derived fibroblasts. Using double-label procedures in which solubilized m e m b r a n e material from contrasting cell lines are mixed and run together on the s a m e gels, it is p o s s i b l e to tell w h e t h e r the same m e m b r a n e components are present in similar or widely different a m o u n t s . T h e d i s t r i b u t i o n o f r a d i o a c t i v i t y along a number of representative gels is shown in Figs. 1-3.

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Plasma membrane components o f skin fibroblasts

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Fig. 1, for example, shows the results of an experim e n t with L-leucine in which the m e m b r a n e s from N cells had been labeled with 3H, while the CF cells had received 14C. The distribution of radioactivity corresponded fairly closely with the staining pattern. The four major protein bands are clearly seen as highly radioactive regions approximately one third the Way along the gel. These bands are not completely resolved from each other, because of the finite (0.8 mm) size of the gel samples. The profiles for 3H and t4c are strikingly similar and coincide closely in every region of the gel. It appears that both sets of membranes contain the same polypeptide components in almost identical amounts. Fig. 2 is an example of the radioactive profiles obtained with gtucosamine-labeled m e m b r a n e preparations9 In this case, CF and HZ lines were compared although l o t h e r c o m b i n a t i o n s gave similar results 9 Again, there was no marked divergence of the 14C and 3H profiles. The coincidence is perhaps not as striking as with the experiments with labeled leucine, probably because of the lower amounts of radioactivity incorpor-

ated into the m e m b r a n e s and the consequent counting variability. Most of the radioactivity was recovered in two regions corresponding with the molecular weight ranges 110,000-90,000 and 60,000-40,000. The material o f low m o l e c u l a r weight in the r e g i o n is p r o b a b l y glycolipid. In Fig. 3, we have compared the 14C and 3H profiles for N and CF m e m b r a n e preparations labeled with Lfucose. Again, the same glycoproteins are present in seemingly identical proportions in the contrasting cell lines. The distribution of radioactivity along the gel observed with fucose is very similar to that seen with glucosamine, indicating that isotope from both compounds is probably incorporated largely into the same macromolecules. DISCUSSION In this study, in contrast to others, 15 we have been unable to detect any major qualitative or quantitative a b n o r m a l i t y in any o f the p r o t e i n or g l y c o p r o t e i n species present in the plasma m e m b r a n e s of fibroblasts

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derived from patients with CF, although a minor c h a n g e such as a single a m i n o acid substitution in one or more c o m p o n e n t s cannot, of course, be ruled out. Such a c h a n g e m i g h t have a crucial effect, for e x a m p l e , on the properties of a surface e n z y m e , or on the stability of the whole m e m b r a n e , yet would not be d e t e c t e d bY our techniques. N e i t h e r w o u l d we be able to detect subtle changes in carbohydrate composition, particularly in those products that were not p r o d u c e d as a result of the m e t a b o l i s m of L-fucose or D-glucosamine. N e v e r t h e l e s s , it seems fair to interpret that the plasma m e m b r a n e of the C F fibroblast is not grossly a b n o r m a l in its protein or glycoprotein constitution. The authors thank Drs. O. M. Rennert, R. Julius, and W. C. Kelly for obtaining the biopsy specimens and initial cultures. REFERENCES

1. di Sant'Agnese PA, and Talamo RC: Pathogenesis and physiopathology of cystic fibrosis of the pancreas, N Engl J Meal 277:1287, 1967. 2. Lobeck CC Cystic fibrosis, in Stanbury JB, Wyngaarden JB, and Fredrickson DS, editors: The metabolic basis of inherited disease, New York, 1972, McGraw-Hill Book Company, Inc. pp 1605-1626. 3. Gugler E, Pallavicini EJ, Swerflow H, and di Sant'Agnese PA: The role of Calcium in submaxillary saliva of patients with cystic fibrosis, J PEDIATR71:585, 1967. 4. Gibson LE, Matthews WJ, Man!han PJ, and Patti JA: Relating mucous, calcium and sweat in a new concept of cystic fibrosis, Pediatrics 48:695, 1971. 5. Bartman J, Wiesmann J, and Blanc WA: Ultrastructure of cultivated fibroblasts in cystic fibrosis of the pancreas, J PEDIATR76:430, 1970. 6. Danes BS, and Bearn AG: Cystic fibrosis: Distribution of mucopolysaccharides in fibroblast cultures, Biochem Bioplays Res Commun 36:919, I969.

The Journal of Pediatrics January 1975

7. Danes BS and Bearn AG: Cystic fibrosis of the pancreas, J Exp Med 129:775 (1969). 8. Danes BS, Foley KM, Dillen SD, and Bearn AG: Genetic study of cystic fibrosis of the pancreas using white blood cell cultures, Nature 222:685, 1969. 9. Fitzpatrick DF, Landon EJ, and James J: Serum binding of calcium and the red cell membrane in cystic fibrosis, Nature New Biol. 235:173, 1972. 10. Matalon R, and Dorfman A: Acid mucopolysaccharides in cultured fibroblasts of cystic fibrosis of the pancreas, Biochem Biophys Res Comrnun 33:954, 1968. 11. Nadler HL, Swae MA, Wodnicki JM, and O'Flynn ME: Cultivated amniotic fluid cells and fibroblasts derived from families, with cystic fibrosis, Lancet 2:84, 1969. 12. Pallavicini JC, Wiesmann V, Uhlendorf WB, and di Sant'Agnese PA: Glycogen content of tissue culture fibroblasts from patients with cystic fibrosis and other heritable disorders, J PEDIATR77:280, 1970. 13. Rennert OM, Frias J, and LaPointe D: Methylation of RNA acid polyamine metabolism in cystic fibrosis, in Mangos JA, and Talamo RC, editors: Fundamental problems of cystic fibrosis and related diseases, New York, 1973, Intercontinental Medical Book Corporation, pp 41-52. 14. Rennert OM, Frias JL, Julius RL, and LaPointe D: The detection of the heterozygote and 'homozygote in cystic fibrosis by short term lymphocyte culture studies: A defect in RNA methylation, Clin Pediatr 11:351, 1972. 15. Pitot H, Benke BJ; and Quissell D: Studies on cystic fibrosis of the pancreas, in Proceedings of gymposium on "Molecular basis of human genetic disease," Gatlinburg, Tenn. 1972, pp. 14-15. 16. Barland P, and Schroeder BA: A new rapid method for the isolation of surface membranes from tissue culture cells, J Cell Biol 45:662, 1970. 17. Weber K, and Osborn M: The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis, J Biol Chem 244:4406, 1969. 18. Turner JC: Triton X-100 scintillant for carbon-14 labeled materials, Int J Appl Radiat Isotop 19:557, 1968.