Lysyl-Protocollagen Hydroxylase Deficiency in Fibroblasts from Siblings with Hydroxylysine-Deficient Collagen

Proc. Nat. Acad. Sci. USA Vol. 69, No. 10, pp. 2899-2903, October 1972 Lysyl-Protocollagen Hydroxylase Deficiency in Fibroblasts from Siblings with Hydroxylysine-Deficient Collagen (prolyl-protocollagen hydroxylase/connective tissue/inborn error/crosslinks) S. M. KRANE, S. R. PINNELL, AND R. W. ERBE Departments of Medicine, Dermatology, and Pediatrics, Harvard Medical School and the Medical, Dermatology, and Children's Services, Massachusetts General Hospital, Boston, Massachusetts 02114 Communicated by E. R. Blout, July 31, 1972 ABSTRACT Cell culture studies were performed on were normal. The hydroxylysine content of dermis was also members of a family in which two sisters, ages 9 and 12, normal in three patients, each with the Marfan and Ehlers- have a similar disorder characterized clinically by severe scoliosis, joint laxity and recurrent dislocations, hyper- Danlos syndromes. Collagen from the skin of the affected extensible skin, and thin scars. The skin collagen from the children was more soluble in denaturing solvents than that sisters was markedly deficient in hydroxylysine, but other derived from controls (4), consistent with a defect in cross- amino acids were present in normal amounts. Hydroxy- linking of collagen molecules, a process in which hydroxylysine lysine in collagen from fascia and bone was reduced to a to involved lesser extent. Since the most likely explanation for the has been thought be critically (2, 5-8). Hydroxyly- hydroxylysihie deficiency was a reduction in enzymatic sine per se is not used in collagen biosynthesis; specific lysyl hydroxylation of lysine residues in protocollagen, we mea- residues are hydroxylated after their incorporation into the sured the activity of lysyl-protocollagen hydroxylase in polypeptide chains of protocollagen (9-12). We, therefore, crude lysates of cultured skin fibroblasts. Enzyme activities postulated that the basis of this disorder was a deficiency in in the two affected children were 14 and 10% of controls, whereas the activity was about 60% of normal in the the enzymatic hydroxylation of lysine. However, lysyl-proto- mother, a pattern most consistent with autosomal reces- collagen hydroxylase could not be adequately measured either sive inheritance. The mutant enzyme demonstrated the in serum or directly in homogenates of the small amounts of same cofactor requirements as that from normal cells. skin available. Deficiency of lysyl-protocollagen hydroxylase is the first of inborn error of human collagen metabolism to be defined In the present investigations, the activity lysyl-proto- at the biochemical level. collagen hydroxylase measured in lysates of cultured skin fibroblasts from the affected siblings was markedly decreased. Heritable disorders of connective tissue include both domi- Skin fibroblast culture was shown to be a convenient system nantly and recessively inherited syndromes (1). Abnormalities for further characterization of the mutant enzyme. in the structural protein, collagen, have been suggested in autosomal, dominantly inherited osteogenesis imperfecta, and MATERIALS AND METHODS in the Marfan and Ehlers-Danlos syndromes (2), but none of Preparation of Fibroblast Lysates. Fibroblasts from biopsy the putative biochemical defects has been established as samples of skin were grown in modified Eagle's medium plus specific for any of these disorders. Autosomal, recessively nonessential amino acids (Gibco) containing 15% fetal-calf inherited homocystinuria due to deficiency of cystathionine serum (Gray Industries, Inc.) and harvested by trypsinization synthetase shares many of the clinical features of the Marfan 48 hr after reaching confluence. Cells were collected by cen- syndrome and produces a secondary defect in connective tissue trifugation at 1200 X g, washed twice by suspension in phos- (3). A distinctive mendelian inheritance pattern provides phate-buffered saline (pH 7.2), and collected by centrifuga- powerful evidence of genetic heterogeneity. We have recently tion. The washed cell pellet was suspended at a density studied a family in which two sisters had some clinical fea- of 108 cells per ml in 0.25 M sucrose, 40 mM Tris * HCl (pH tures of the Ehlers-Danlos and Marfan syndromes but in which 7.4), and 50 mM 2-mercaptoethanol, and the cells were lysed both parents and an older sister were unaffected, suggesting by sonication for 2 min in a Raytheon model DF101 sonic recessive inheritance (4). The affected 9- and 12-year old oscillator. The crude lysate was clarified by centrifugation at sisters had severe progressive scoliosis present since infancy, 50,000 X g for 15 min. hyperextensible skin, marked joint laxity and recurrent joint Fibroblasts were cultured from the skin of affected and un- dislocations, and mild arachnodactyly. Ectopia lentis was affected members of the family with hydroxylysine-deficient absent, intelligence was normal, and urinary amino acids were collagen (4). Patient 1 refers to the proposita, a 12-year-old not increased. Amino-acid analysis of dermal collagen from girl, and Patient 2 to her affected 9-year-old sister. Cells were the affected sisters disclosed a marked reduction in hydroxyly- also cultured from the 16-year-old unaffected sister, and from sine to about 5% of normal, but normal amounts of hydroxy- the father and mother. Cell cultures from normal controls proline and other amino acids. The hydroxylysine content age-matched to each family member were grown under identi- was also reduced in samples of lumbodorsal fascia and variably cal conditions, and lysates were similarly prepared. reduced in bone, but was about normal in a single sample of costal cartilage. The amino-acid compositions of skin from Preparation of Protocollagen Substrates. Labeled lysyl-proto- the parents and from the clinically unaffected older sister collagen was prepared by incubation of 50-85 cartilagenous 2899 Downloaded by guest on September 26, 2021 2900 Medical Sciences: Krane et al. Proc. Nat. Acad. Sci. FSA 69 (1972) X 106 dpm; 14C, 0.5-2.0 X 105 dpm), 25 mM Tris-HCl (pH ZI) 7.2), 0.1 mM a-ketoglutarate, 0.1 mM FeSO4, 0.5 mM ascorbic CONTROL acid, 0.1 mM dithiothreitol, 2 mg bovine serum albumin, 0.5 mg catalase, and solution of the enzyme to be tested. The reaction was usually initiated by addition of substrate, and samples were incubated at 370 for various periods, usually 90 min. The reaction was terminated by addition of 10 ml of cold :I (-20°) acetone (15). Reaction mixtures were kept at -20° for at least 30 min and centrifuged at 3000 X g for 30 min. The supernatant solution was then aspirated. The pellets 2 were dried in a gentle stream of nitrogen and suspended in 10 ml of 0.3 M citrate-phosphate buffer, pH 6.4. 14C- or 3H- labeled hydroxylysine was measured by oxidizing the MINUTES terminal carbon to formaldehyde with periodate, coupling FIG. 1. Lysyl-protocollagen hydroxylase activity in cells of with dimedone, and extracting the adduct with 15 ml of the 9-year-old girl with hydroxylysine-deficiency (Patient 2) com- toluene by the method of Blumenkrantz and Prockop (17). pared with age-matched normal control. Formation of [3H]- Radioactivity in 10 ml of the toluene phase was measured hydroxylysine is plotted as a function of time and normalized with a liquid scintillation spectrometer in counting vials to an equivalent number of cells in the lysates. containing 2 ml of 1.5% 2,5-diphenyloxazole and 0.005% p-bis [2-(5-phenyloxazolyl) ]-benzene in toluene, at an effi- for in ml of Krebs ciency of about 49% for 3H and about 87% "4C. tibiae from 10- to 12-day-old chick embryos 3.0 In some experiments we assayed formation of labeled hy- "medium A" (13) containing 1 mM a,a'-dipyridyl for 30 min droxylysine by stopping the reaction with HCl in a final con- at 370 (14,15). Then, either 1251sCiofL[U-14C]lysine (250 Ci/ centration of 6 N, hydrolyzing for 20 hr at 1080, adding car- mol) or 400 ,uCi of generally labeled L- [8H ]lysine (3 Ci/mmol) rier hydroxylysine, drying under reduced pressure, placing the L- were added, and or 400 ,Ci of [4,5-'H]lysine (27 Ci/mmol) sample on the column of the automatic amino-acid analyzer the incubation was continued for 2 hr. The tibiae were re- without with (18), and collecting the entire effluent in fractions moved and homogenized at 20, in 8 ml of distilled water with ninhydrin. The fractions containing hydroxyly- at X reacting a Teflon and glass homogenizer, and centrifuged 100,000 sine were identified colorimetrically (19), and aliquots were g for 1 hr. The supernatant solution was dialyzed against counted in a thixotropic gel containing 4% Cab-O-Sil (G.L. several changes of 20 mM Tris HCl (pH 7.6) at 2-4°, then Cabot, Inc.) in Instagel (Packard Instrument Co.). In other heated in a boiling water bath for 10 min and cooled. The sub- Labeled experiments, formation of hydroxylysine was assayed by strate was stored at -200 in 0.5-ml portions. prolyl- measurement of the exchange of 3H with H20 after hydroxyla- protocollagen was prepared as above with 50 ,Ci of L-[U-14C]- tion (20). Lysyl-protocollagen substrate labeled with [4,5- proline (192 Ci/mol) or 400 pCi of 143,4-'H]proline (4.8 Ci/ En- 3H]lysine was used in these assays. The reaction was stopped mmol). Labeled amino acids were purchased from New by addition of trichloroacetic acid in a final concentration of gland Nuclear Corporation. 15%, and the supernatant solution was separated by centrif- Enzyme Assays. The incubation mixture used for assay of ugation at 3000 X g. The supernatant was then distilled lysyl-protocollagen hydroxylase was similar to that described under reduced pressure, and '0.5 ml of the distillate was for prolyl-protocollagen hydroxylase (16) and contained, in a counted in the thixotropic gel. volume of 1.0 ml, lysyl-protocollagen substrate (8H, 0.5-2.0 Most of the assays for lysyl-protocollagen hydroxylase ,CONTROL IC .44 .4i ,PATIENT 2 0 1 2 3 ['c]7L )SINE SuBSTRATE dpM 10-5 *2 FIG.

Load more