Abnormal Properties of Collagen Lysyl Hydroxylase from Skin Fibroblasts of Siblings with Hydroxylysine-Deficient Collagen
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Abnormal Properties of Collagen Lysyl Hydroxylase from Skin Fibroblasts of Siblings with Hydroxylysine-Deficient Collagen Rum S. QuiN and STEPH M. KRANE From the Department of Medicine, Harvard Medical School, and Medical Services (Arthritis Unit), Massachusetts General Hospital, Boston, Massachusetts 02114 A B S T R A C T Skin fibroblasts from two siblings with part due to differences in cofactor concentrations and in hydroxylysine-deficient collagen (Ehlers-Danlos syn- rate and sequence of events in collagen synthesis in dif- drome, type VI) contained normal levels of collagen ferent tissues. prolyl hydroxylase activity but were markedly deficient in collagen lysyl hydroxylase activity. The deficiency was evident in all fractions of cell lysates, in low and INTRODUCTION high ionic strength buffers, and in detergent. Assays of Current knowledge of the events in collagen metabolism mixtures of wild-type and mutant cell lysates indicated is such that the biochemical basis of certain connective no activation of mutant enzyme by factors in wild-type tissue disorders can now be explained. Human hydroxy- cells or inhibition of normal enzyme by material in mu- lysine-deficient collagen disease (Ehlers-Danlos syn- tant cells. Wild type or mutant cells cultured with as- drome, type VI) is a disorder characterized by a spe- corbic acid (50 ug/ml of culture medium, added daily) cific aberration in collagen synthesis. This heritable dis- contained approximately the same level of lysyl hy- ease was first identified in two sisters with features sug- droxylase activity as cells cultured without ascorbate, gestive of both Ehlers-Danlos and Marfan syndromes but prolyl hydroxylase activity without ascorbate (1). The clinical manifestations of the disease included was depressed in both an average of 41%. The mutant kyphoscoliosis, joint laxity with recurrent joint disloca- lysyl hydroxylase was less stable at 370C than the wild tions, hyperextensible skin, and slow wound healing. type and did not form high molecular weight aggregates Amino acid analysis of the dermis from the two children in low ionic strength buffers, as did the control enzyme. showed that the skin collagen contained a normal amount The activity of the mutant enzyme was maximally stim- of hydroxyproline, but the hydroxylysine content was ulated after dialysis against buffer solutions containing markedly reduced to approximately 5% of normal. The 10 mM dithiothreitol. When assayed in 100 iAM dithio- hydroxylysine content of other tissues was reduced to a threitol, the mutant enzyme exhibited a higher apparent variable extent; fascia contained 14-28%, bone about Km for ascorbate (20 /AM) than the wild type (4 /AM). 43%, and cartilage 90% of the amount present in corre- In 1.0 mM dithiothreitol the mutant enzyme's apparent sponding tissues from normal subjects. The skin collagen Km for ascorbate was reduced to 5 aM. Wild type and from these patients was abnormally soluble in denaturing mutant enzymes had the same apparent Km for a-keto- solvents such as 4 M CaCl2 and 9 M KSCN. These glutarate (20,sM). The properties of prolyl hydroxylase observations were consistent with a deficiency of hy- in wild type and mutant cells were identical: apparent droxylysine-derived intermolecular cross-links, and Km's for ascorbate and a-ketoglutarate were 100 /AM and analysis of borohydride-reducible cross-links indicated 20 /AM, respectively. If mutant enzyme protein with al- abnormalities in the nature, distribution, and amount of tered kinetic properties is the only enzyme functioning these moieties in the dermal collagen (2). to hydroxylate lysyl residues in collagen, the variations Hydroxyproline and hydroxylysine are formed by en- in hydroxylysine content observed in collagen from dif- zymatic oxidation of prolyl and lysyl residues in pre- ferent tissues in the subjects reported here could be in cursor component chains of the collagen molecule. The Received for publication 30 June 1975 and in revised form hydroxylations are catalyzed by two separate enzymes, 25 August 1975. prolyl hydroxylase, (prolyl-protocollagen hydroxylase The Journal of Clinical Investigation Volume 57 January 1976-83-93 83 or collagen prolyl hydroxylase EC 1.14.11.2), and lysyl pension was lysed by sonication for 2 min in a Raytheon hydroxylase (lysyl-protocollagen hydroxylase or colla- model DF 101 sonic oscillator (Raytheon Co., Sorensen gen lysyl hydroxylase). Lysates of cultured skin fibro- Operation, Production Equipment Dept., Norwalk, Conn.). The lysate was centrifuged at 50,000 g for 60 min, and the blasts from the affected siblings contained normal levels supernate was carefully separated from the overlying lipid of prolyl hydroxylase activity but reduced lysyl hydroxy- layer. In some experiments acetone powders were prepared lase activity (less than 15% of that present in fibroblasts according to the method of Morton (13). from normal subjects) (3). In vitro assays of Protocollagen substrate. The method of preparation of prolyl [3,4-'H]proline or [G-'H]lysine (New England Nuclear, and lysyl hydroxylase activity in crude lysates of normal Boston, Mass.) protocollagen substrate from chick embryo and mutant cells require the presence of oxygen, ca-keto- tibia (4, 11) as modified by Krane et al. (3) was used. glutarate, ascorbic acid, and ferrous ion for the hy- Although the same method of substrate preparation was droxylation reactions to occur at optimal rates. Thus the always followed, for unexplained reasons different substrate preparations exhibited variable activities with the same enzymes in human skin fibroblasts have characteristics enzyme preparation. in common with prolyl hydroxylases from human and rat With constant amounts of control human fibroblast lysate liver (4, 5), newborn rat skin (6), chick embryos (7), and incubation times of 60-120 min, a plateau in hydroxyly- and mouse L-929 fibroblasts (8, 9), as well as lysyl hy- sine formed was not reached with amounts of underhy- droxylated collagen substrates tested up to 10 times those droxylases from chick embryo (10, 11) and fetal rat skin used in most assays reported here. Because of the amounts (12). of radioactivity necessary and the relatively low yields of Further studies of collagen hydroxylases in skin fibro- underhydroxylated substrate, it was not practical to deter- blasts were performed to learn more about human en- mine saturating conditions for every substrate preparation zymes from or to attempt to employ saturating collagen substrate con- this source. Comparison of the properties ditions in every assay. of hydroxylases from normal and deficient cells might Enzyme assays. Assays for lysyl or prolyl hydroxylase thus help to elucidate the cause of defective collagen contained (in a total volume of 1.0 ml) 25 mM Tris-HCl lysyl hydroxylation. This communication reports studies (pH 7.2), 100 gtM a-ketoglutarate (Sigma Chemical Co., of characteristics of collagen lysyl and prolyl hydroxy- Inc., St. Louis, Mo.), 100 ,uM ferrous sulfate, 500 pM ascorbic acid (recrystallized, Fisher Scientific Co., Pitts- lases in normal cells and in cells from two siblings with burgh, Penn.), 0.5 mg catalase (Sigma), 100 /LM dithio- hydroxylysine-deficient collagen (3). The lysyl hydroxy- threitol (DTT) (Calbiochem, San Diego, Calif.), 2 mg of lase from deficient cells has chemical, physical, and bovine serum albumin (Miles Laboratories, Inc., Elkhart, kinetic properties significantly different from the enzyme Ind.) cell lysate (usually 10-25 1ul of lysates from sonicated in normal cells. cells suspended at 0.1-1 X 10' cells/ml) and 0.5-2.5 X 10' dpm of protocollagen substrate. The enzyme reactions were METHODS initiated by addition of either substrate or enzyme and, unless otherwise indicated, assay mixtures were incubated Fibroblast lysates. Cells were grown in 100-mm diameter for 60-120 min in air at 37°C. Prolyl hydroxylase assays plastic petri dishes (Falcon Plastics, Division of BioQuest, were terminated by the addition of 0.5 ml of 15% trichloro- Oxnard, Calif.) or in glass roller bottles, the latter with acetic acid. Tritiated water produced on hydroxylation of an approximate growth surface area of 600 cm'. Mutant 1 labeled proline was separated from the reaction mixture and 2 refer to cells grown from patients 1 and 2, respec- by vacuum distillation. Portions of the distillate were tively, with Ehlers-Danlos syndrome type VI (1, 3). Most counted in 10 ml of Insta-gel (Packard Instrument Co., Inc., cultures were in passage 10-20. Control cells used in earlier Downers Grove, Ill.) containing 3 ml of H20 with an studies were from normal subjects age-matched for the efficiency of approximately 30%. Lysyl hydroxylase assays mutant subjects. Some of these cells were no longer avail- were terminated by addition of 10 ml of cold (-20°C) able during the present studies. All control cells used in the acetone. The labeled formaldehyde produced by periodate present experiments were from children aged 3 wk-18 yr oxidation of carbon 6 of [G-'H]hydroxylysine was coupled and matched approximately for passage number and growth with dimedone, and the complex was extracted into 15-20 rate. The culture medium, modified Eagle's medium (Grand ml of toluene (3, 14). 10 ml of the toluene phase was added Island Biological Co., Grand Island, N. Y.) supplemented with to a scintillation counting vial containing 2 ml of 1.5% 2,5 15%o fetal calf serum, glutamine, essential and nonessential diphenyloxazole and 0.005%o p-bis [2- (5-phenyloxazolyl)] - amino acids, glucose