A [3H]lysine-containing synthetic peptide substrate for human protocollagen lysyl hydroxylase

Biochimica et Biophysica Acta 840 (1985) 143-152 Elsevier

143

BBA 22044

A [3Hllysine-containing synthetic peptide substrate for human protocollagen

lysyi hydroxylase David B. Glass *, Philip P. Dembure, Jean H. Priest and Louis J. Elsas, II Department of Pharmacology and Division of Medical Genetics, Department of Pediatrics, EmoO, Unit?ersity School of Medicine, Atlanta, GA 30322 (U.S.A,) (Received October 22nd, 1984) (Revised manuscript received February 13th, 1985)

Key words: Lysine; Peptide substrate; Lysyl hydroxylase; Protocollagen; (Human)

A tridecapeptide containing tritium-labelled lysine and corresponding closely to residues 98 to 110 of the a chain of type I collagen was synthesized by the solid-phase method. Giy-Leu-Hyp-Gly-Nle-14,5-3H]Lys-GlyHis-Arg-Gly-Phe-Ser-Gly was used as a substrate of human protocollagen lysyl hydroxylase (peptidyllysine, 2-oxoglutarate: oxygen 5-oxidoreductase, EC 1.14.11.4) obtained from dermal fibroblasts. L-14,5-3H]Lysine was converted to Na-t-butyloxycarbonyI-N~-o-chlorobenzyloxycarbonyil3H]iysine which was incorporated during stepwise synthesis of the peptide. The chemical and radiochemical purities and specific activity of the completed peptide were characterized. A non-radiolabelled analogue of the peptide inhibited the hydroxylation of [3H]lysine-containing protocollagen by human lysyl hydroxylase, indicating that the synthetic peptide interacted with the enzyme. The peptide containing [3H]lysine was a substrate for lysyl hydroxylase and permitted direct measurement of enzyme activity in relatively crude cell extracts by a tritium-release assay. Extracts of cultured fibroblasts from a patient with an autosomai recessive pattern of inheritance for Ehlers-Danlos syndrome type VI had activities for tritium release from either the radiolabeUed synthetic peptide or from [3Hllysine-containing protocollagen that were only 30% of those from control cells. These data indicate that a stable, well-defined synthetic peptide containing [3H]lysine is a useful substrate for studies of genetically variant lysyl hydroxylase from cultured human cells.

Introduction

Since the discovery of impaired activity of protocollagen lysyl hydroxylase (EC 1.14.11.4) in Ehlers-Danlos syndrome type VI [1], the catalytic properties of the mutant enzyme have been studied * To whom correspondence should be addressed. Abbreviations: Boc, t-butyloxycarbonyl; Z, benzyloxycarbonyl; (Hyp1°°, Nlel°2)al(I)(98-110), the tridecapeptide Gly-LeuHyp-Gly-Nle-Lys-Gly-His-Arg-Gly-Phe-Ser-Gly corresponding to residues 98 to 110 in al(I) collagen; (Hyp1°°, Nle 1°2, [3H]Lys1°3)al(1)(98-110), the radiolabelled peptide Gly-LeuHyp-Nle-[4,5- 3H]Lys-His-Arg-Gly-Phe-Ser-Gly corresponding to residues 98 to 110 in al(1) collagen.

by several investigators. Mutant lysyl hydroxylases from patients with similar phenotypes were found to have either an elevated [2] or an unaltered [3,4] K m for ascorbate as compared to that of the normal enzyme. These results suggest either variations in assay conditions among the studies or heterogeneity in the genotypes of the individuals. Since the use of large amounts of ascorbate may improve collagen production in some individuals with Ehlers-Danlos syndrome [5], knowledge of the role of ascorbate in protocollagen lysyl hydroxylation by the mutant enzyme has immediate clinical relevance. Chemically well-defined substrates [6] will be useful in standardizing assays of

0304-4165/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

144

human lysyl hydroxylase and in better understanding cofactor requirements and other mechanisms producing variation in the mutant enzyme from different affected families. Lysine-containing synthetic peptides with amino acid sequences comparable to portions of collagen are substrates for protocollagen lysyl hydroxylase [6-8]. Such synthetic peptides have been useful in studying the reaction catalyzed by normal protocollagen lysyl hydroxylase obtained from nonprimate tissues, primarily chick embryos. The determinants of substrate specificity for hydroxylation of peptidyllysine residues [6,7], the molar activity value of the enzyme [8], the kinetic mechanism of the reaction [9,10], the source of oxygen involved in the hydroxylation [11], and the role of ascorbate as cofactor in both the coupled [9,10] and uncoupled [12] reactions have been elucidated with synthetic peptide substrates. Use of such a synthetic substrate has advantages over assay methods that employ preparations of unhydroxylated protocollagen from avian embryonic bone in either their non-radiolabelled or [14C]lysine- or [3H]lysine-labelled forms [13-17]. These benefits include greater stability and purity of the peptide as compared to crude preparations of protocollagen and availability of peptide such that sufficient concentrations can be used to ensure reasonable linearity of the assay. In addition, synthetic peptides are well-defined substrates with usually a single site of hydroxylation, a fact that makes them useful in mechanistic studies. However, in some instances, assays using synthetic peptide substrates have unique disadvantages. The chemical measurement of hydroxylysine formed in a peptide substrate [14] is rather tedious and insensitive. Assays that utilize c~-keto[14C]glutarate as a cosubstrate and measure the stoichiometric release of 14CO2 which is coupled to peptidyllysine hydroxylation [6,13] are convenient but are most useful for purified or at least partially purified lysyl hydroxylase. In less pure cell extracts, the 14CO2-release assay is relatively non-specific due to the decarboxylation of a-ketoglutarate catalyzed either by the more active protocollagen prolyl hydroxylase (assuming a suitable cosubstrate is present) or by a number of other decarboxylases able to act on c~-ketoglutarate [18]. A convenient assay for the activity of lysyl

hydroxylase in relatively crude extracts of cultured human fibroblasts would be useful in investigations of variants of the Ehlers-Danlos syndrome type VI [3-5,19-21]. Protocollagen lysyl hydroxylase from these patients is most readily studied in crude or only partially purified preparations because it is usually obtained from cultured cells and the levels and activity of the enzyme are low. Normal human lysyl hydroxylase has recently been purified to homogeneity [4], but all reported studies of the mutant enzyme have been performed with crude preparations of both enzyme and protocollagen substrate [1-4]. We report the synthesis and use of Gly-Leu-Hyp-Gly-Nle-[4,5-3H]LysGly-His-Arg-Gly-Phe-Ser-Gly ((Hyp 1°°, Nle m2, [3 H]Lys~03)cd(I)(98-110)), a peptide closely corresponding to amino acid residues 98 to 110 in the cd(I) chain of collagen [22]. This [3H]lysine-containing peptide has the advantages described above for synthetic substrates, is of defined specific radioactivity, and can be used in an analogous fashion to [3H]lysine-labelled protocollagen in which tritium is released to water during the hydroxylation reaction [13,16,17]. Its use for the specific assay of lysyl hydroxylase activity in preparations of cultured dermal fibroblasts from normal individuals and patients with Ehlers-Danlos syndrome is described. The non-radiolabelled form of this peptide has recently been used as a model substrate for the cyclic AMP-dependent protein kinase [23]. Materials and Methods

A nalytic methods Thin-layer chromatography (TLC) was performed on silica gel G sheets with the following solvents: CHCl3/methanol/acetic acid (85 : 10 : 5, v/v); 1-butanol/acetic acid/water (4 : 1 : 1, v/v); and 1-butanol/pyridine/acetic acid/water (15: 10 : 3 : 12, v/v). Microcrystalline cellulose sheets were also used with 1-butanol/pyridine/acetic acid/water (as above) as well as 1-butanol/acetic a c i d / w a t e r ( 4 0 : 6 : 15, v / v ) , 1 - b u t a n o l / isopropanol/monochloroacetic acid/water (65: 15 : 3:20, v / v / w / v ) , and ethyl acetate/pyridine/acetic acid/water (5 : 5 : 1 : 3, v/v). Peptides and amino acids were detected with ninhydrin. For chromatographed N~-Boc-amino acids, one

145 channel was exposed and one replicate channel was not exposed for 15 min to fumes of concentrated HC1 prior to staining. Rv values refer to single spot chromatograms. Amino acid derivatives were co-chromatographed with known standards. For determination of radiochemical purity of compounds, appropriate channels of unstained chromatograms were cut into 1-cm sections and counted in a liquid scintillation spectrometer in a toluene-based fluor [24]. Melting points were determined on a FisherJohns apparatus and are uncorrected. Optical rotations were measured on an O.C. Rudolph polarimeter (2 dm path length). Elemental analyses were performed by Atlantic Microlabs. High-voltage electrophoresis was performed at pH 1.9 on cellulose sheets [25]. Amino acid analysis were performed on replicate acid hydrolysates (5.7 N, ll0°C, 24 h) or enzymic digests (see below) of synthetic peptides using a Beckman model 119CL amino acid analyzer. Results were not corrected for loss of serine. When the hydrosylate of (Hyp 1°°, Nle 1°2, [3H]Lysl°3)al(I)(98-10) was run, fractions of effluent from the analyzer were collected and counted for tritium. Peptides were enzymatically digested in sequence by papain (20: 1, w/w, in 0.1 M ammonium acetate (pH 6.0) and 0.02 M 2mercaptoethanol at 37°C for 6 h) followed by lyophilization and then aminopeptidase M (10:1, w / w , in 0.1 M ammonium bicarbonate (pH 7.8) at 37°C for 18 h). This procedure allowed an assessment of the optical purity of the peptides and whether or not side reactions occurred during synthesis that resulted in acid-labile modification of amino acids which would not be detected on analysis of acid hydrolysates. N "-t -Butyloxycarbonyl-N ~-o-chlorobenzyloxycarbonnyl[~H] lysine

p-Nitrophenyl-o-chlorobenzyl carbonate was synthesized from p-nitrophenyl chloroformate (14.1 g, 0.07 mol) and o-chlorobenzyl alcohol (10.0 g, 0.07 mol) as described by Yamashiro and Li [26] for the preparation of the corresponding pbromobenzyl ester. Yield, 13.5 g (63%); m.p., 74-76°C; TLC on silica gel in CHC13/methanol / acetic acid, R v 0.90. Anal. Calculated for C14H10NOsC1 (307.69): C, 54.65; H, 3.28; N, 4.55;

C1, 11.52. Found: C, 54.58; H, 3.33; N, 4.50; C1, 11.48. N'-o-Chlorobenzyloxycarbonyl[ 3H]lysine was synthesized as described by Yamashiro and Li [26] for the corresponding N~-p-bromo-Z-protected derivative except that p-nitrophenyl-o-chlorobenzyl carbonate was used in place of p-nitrophenyl-pbromobenzyl carbonate. The synthesis was conducted on a 0.25 mmol scale in which 7.84 mCi of c-[4,5-3H]lysine (0.032 mg, 0.174 /zmol) was combined with 45.7 mg (0.25 mmol) of non-radiolabelled lysine and used as starting material. The product was chemically and radiochemically pure as determined by TLC on silica gel in several solvent systems. The yields of both mass and tritium are presented in Results. Other analytical tests were performed on non-radiolabelled N F-ochloro-Z-lysine that was synthesized identically but on a larger (3.0 mmol) scale. Yield, 90%; m.p. 229-231°C; TLC on silica gel in butanol/acetic acid/water, R v 0.45 and butanol/pyridine/acetic acid/water, R F 0.54; [c~]~4 + 19.5°C (c = 0.6, 80% acetic acid). Anal. Calculated for C~4HIgN:O4C1 (314.77): C, 53.42; H, 6.08; N, 8.90; Cl, 11.26. Found: C, 53.37; H, 6.11; N, 8.88; C1, 11.19. The product contained no N", N~-di-(o-chloro-Z)[3H]iysine could be quantitatively reconverted to lysine by catalytic hydrogenation. N " - t - B u t y l o x y c a r b o n y l - N ~-o-chlorobenzyloxycarbonyl[3H]lysine was prepared from the N~-o chloro-Z-[3H]lysine by reaction with 2-Boc-oxyimino-2-phenylacetonitrile (77 mg, 0.31 mmol) as described by Itoh et al. [27]. The product was not crystallized but was obtained as an oily residue by rotoevaporation of the ethyl acetate extract. Yields of mass and tritium of the radiolabelled product were determined (see Results). Other analytical tests were performed on non-radiolabelled material that was synthesized on a larger (0.5 mmol) scale. Yield, 187 mg (90%); TLC on silica gel in chloroform/methanol/acetic acid, Rv 0.76; butanol/ acetic acid/water, Rv 0.66; and b u t a n o l / pyridine/acetic acid/water, R v 0.63. These R F values were identical to those of the standard N"-Boc-N~-o-chloro-Z-lysine. The product was radiochemically pure in these solvent systems. Substrate preparation

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146 Lysl°3)M(I)(98-110) and its non-radiolabelled analog (Hyp 1°°, Nlel°2)M(I)(98-110) were synthesized manually by solid phase techniques [28,29] using a double-coupling protocol described previously [30,31]. The following N~-Boc-amino acids were used; Gly, Ser(O-benzyl), Phe, Arg(N (;NO2) , His(Nim-tosyl), Lys(N~-o-chloro-Z), Nle, Hyp(O-benzyl), and L e u . H20. Dicyclohexylcarbodiimide was used as the coupling agent. Completeness of the coupling of each residue was assessed by the ninhydrin test [32]. Tritium-labelled and corresponding non-radiolabelled peptides were synthesized in a coordinated manner. The peptide was prepared on a 1.0 mmol scale using 1% crosslinked chloromethylated styrene-divinylbenzene resin that had been esterified (0.39 retool/g) to N"-Boc-Gly. After the peptide was synthesized through glycine-104, the resin was dried and an amount equivalent to 0.2 mmol of peptide was removed. Non-radiolabelled N%Boc-N'-o-chloro Z-lysine was then used to completed the synthesis of the remaining 0.8 mmol of peptide. (Hyp l°°, Nle 1°2, [3H]Lysl°3)al(I)(98-110) was synthesized using N%Boc-N~-o-chloro-Z-[3 H]lysine and the 0.2 mmol of peptidyl resin removed from the starting material. Completed peptides were cleaved from the resin with HBr in trifluoroacetic acid in the presence of a 50-fold molar excess of anisole for 90 min and were deprotected by catalytic hydrogenation in 90% acetic acid for 24 h [33]. Peptides were purified by cation-exchange chromatography [34]. The peak of peptide material was pooled, lyophylized, and subjected to partition chromatography on a 1.5 × 90 cm column of Sephadex G-25 (fine) [35] using a solvent system of butanol/pyridine/0.1% acetic acid (5 : 3 : 11, v/v) [36]. Peptide material in column effluents was detected with fluorescamine [37]. The major peak was pooled, rotoevaporated, and lyophilized from water. Yield of purified portion of (Hypl°°, Nle 1°2, [3H]Lysl°3)al(I)(98-110), 33 mg (13% based on starting resin). Amino acid analysis of acid hydrolysate: Hyp (0.97), Ser (0.93), Gly (4.85), Leu (1.05), Nle (1.08), Phe (1.07), Lys (1.10), His (0.95), Arg (1.02); of enzymic digest: Hyp (0.96), Ser (0.89), Gly (5.02), Leu (0.89), Nle (1.20), Phe (1.14), Lys (0.92), His (0.94), Arg (1.04). The composition of the corresponding non-radiolabelled peptide has been published previously [23].

TLC on cellulose in butanol/pyridine/acetic acid/water, R F 0.53; butanol/acetic acid/water, R v 0.23; butanol/isopropanol/monochloroacetic acid/water, R v 0.17; and ethyl acetate/pyridine/ acetic acid/water, Rv 0.16. High-voltage electrophoresis R v 0.77 compared to lysine standard (R v 1.00). All R v values were identical for the tritium-labelled and non-radiolabelled peptides. Crude preparations of unhydroxylated protocollagen labelled with [3H]lysine were prepared from 14-day-old chick calvaria as previously described [3,38]. The specific activities of those preparations were 5.5 to 8.0 t~Ci/mg protein. Enzyme preparation

Human skin fibroblasts from a patient with Ehlers-Danlos syndrome type VI [38] and from clinically normal individuals were cultured in monolayer using Dulbecco and Vogt's medium supplemented with 15% fetal bovine serum and non-essential amino acids as previously described [39]. Lysyl hydroxylase was extracted from cells as described by Kivirikko and Myllylg [13]. Confluent cells from a 75 cm z flask were washed twice with phosphate-buffered saline (pH 7.4) and resuspended in ice-cold 20 mM Tris-HCl (pH 7.5) containing 0.2 M NaCI, 0.1 M glycine, 0.05 mM dithiothreitol, and 0.1% Triton X-100 to give a concentration of 1.5-2.5 mg protein/ml (0.75 ml buffer per 75 cm: flask of cells). The cells were disrupted by sonification for 10 s at a setting of 40 (Fisher Sonic Dismembrator) and the resulting extract was centrifuged at 15 000 × g for 10 min. The supernatant fraction was used immediately for enzyme assays without further treatment to remove any collagenous materials present. Protein was determined by the method of Lowry et al. [40] with bovine serum albumin as standard. Lysyl hydroxylase assay

Lysyl hydroxylase activity was assayed in a total volume of 0.5 ml containing either 0.5 mM (Hyp 1°°, Nle 1°2, [3H]Lysl°3)o~1(I)(98-110) or [3H]lysine_containing protocollagen (approx. 300000 dpm) and 20 mM Tris-HCl (pH 7.5), 0.5 mM ascorbate, 0.1 mM dithiothreitol, 0.5 mM a-ketogluterate, 0.05 mM FeSO4, 1.5 m g / m l bovine serum albumin, 0.5 m g / m l catalase and 0.10-0.15 mg protein from the enzyme prepara-

147

tions. The reaction was initiated by addition of radiolabelled substrate and conducted at 37°C for 60 min or for the indicated times. Enzyme activity using either of the radiolabelled substrates was determined by the tritium-release method of Peterkofsky and DiBlasio [17] as previously modified [3,38]. The supernatant fractions of acidified, deproteinated reaction mixtures were passed over ion-exchange columns (Dowex 50-X8) which removed excess radiolabelled peptide or protein substrate and any free [3H]lysine that might have been generated during the assay. The [3H]water in the column effluents was determined by liquid scintillation spectroscopy at an efficiency of 35%. All assays were conducted in triplicate unless otherwise specified. Materials L-[4,5-3H]Lysine (45-50 C i / m m o l ) was purchased from ICN. Upon lyophilization, 9.9% of the tritium was lost. The remaining material was pure by both TLC and high-voltage electrophoresis. o-Chlorobenzyl alcohol, p-nitrophenyl chloroformate, and 2-Boc-oxyimino-2-phenylacetonitrile were from Aldrich Chemical Co. Dicyclohexylcarbodiimide was purchased from Pierce Chemical Co. N%Boc-amino acids were from Peninsula Laboratories. Standard N~-o-chloro-Z-lysine was prepared by treatment of N%Boc-NLo-chloro-Zlysine with 33% trifluoroacetic acid in methylene chloride for 45 min at room temp. Ascorbic acid, a-ketoglutarate and catalase were purchased from Calbiochem. Lysine HC1, bovine serum albumin and dithiothreitol were from Sigma, and Dowex 50-X8 was from Bio-Rad.

Results The crude peptide material from the solid-phase synthesis contained several components. The major peak from the SP-Sephadex column (Fig. 1A) was further resolved into four peaks on partition chromatography (Fig. 1B). The major partition chromatography peak with R E 0.24 was (Hyp1°°, Nle 1°2, [3H]Lysl°3)a1(I)(98-110), as confirmed by the analytic data (see Materials and Methods). Additionally, the ultraviolet Spectrum of the peptide indicated that no NG-nitroarginine was present and that the phenylalanyl residue had not been

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converted to fl-cyclohexylalanine (data not shown). The peak of R E 0.15 had the amino acid composition of Ser (0.95), Gly (2.96), Phe (1.04), Lys (1.06), His (0.94), Arg (1.05) and was of higher specific activity than that of the desired product. It was ([3H]Lysl°3)td(I)(103-110), a peptide prematurely terminated after the incorporation of N"Boc-N~-o-chloro-Z-[aH]lysine. The minor peaks with R F values of 0.60 and 0.31 were side products in which the seryl residue had been damaged by alkylation or bromination. The final product was radiochemically pure as determined by TLC on cellulose in butanol/pyridine/acetic acid/

148

water and by high-voltage electrophoresis (Fig. 2). All of the tritium in a hydrolysate of the peptide was present as [3H]lysine, as determined by recovery of tritium from the amino acid analyzer (data not shown). Lyophilization of the final product did not remove any tritium as [3H]water. The recoveries of lysine and tritium during the synthesis of (Hyp m°, Nle m2, [3H]Lysm3)M(I)(98 110) from [3H]lysine were similar through the first three steps (Table I). The yields at each of steps 2 - 4 were 50-60%. The largest loss of tritium occurred in step 5, cleavage of the peptide from the resin and its deprotection and purification. Only 10% recovery overall was obtained in these procedures. The specific activity of the final product was 5.8 mCi/mmol.

TABLE I S Y N T H E S I S O F I . - [ 3 H ] L Y S I N E - C O N T A 1 N I N G COLL A G E N PEPTIDE F R O M L-[3H]LYSINE The yields of material and radioactivity were quantitated at each step in the synthesis of (Hyp 1°°, Nle m2, [3H]Lys m3) cd(1)(98-10) from [3H]lysine. The numbers in parentheses indicate recoveries (%) based on either the mass or radioactivity of the starting material. Each compound was synthesized as described in Materials and Methods. The sequence below corresponds to amino acid residues 98 to 110 in the cd(l) chain of chick (and rat and calf) collagen [22]. GlygS_Leu_Pro_Gly_Met_Lys_Gly_His_Arg_Gly_Phe_Ser_Gly~ 1o Step

Compound

Amount of compound (/~mol)

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1 2 3

1,-[-~H]lysine NLo-CIZ-L-[3HJlysine N"-Boc-N'-o-CIZ-L [3H]lysine (Hyp II~l, Nle ~(12, [3H]Lys 1°3 ) a1(1)(98-110), solid-phase ~' (Hyp 1{~},Nle ill2, [3H]Lys 1113)

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The ability of the non-radiolabelled peptide, (Hyp t°°, Nlem2)~l(I)(98-110), to inhibit the hydroxylation of lysine residues in protocollagen by normal human fibroblast lysyl hydroxylase was examined at two fixed concentrations of unhydroxylated, radiolabelled protocollagen substrate. The protein concentrations of the protocollagen preparation were chosen to be above and below the apparent K m value of the enzyme for this substrate which was approx. 50 /~g protein/ml (data not shown). Increasing amounts of the synthetic collagen peptide inhibited the enzyme activity in a concentration-dependent manner (Fig. 3). In each case, 2 mM peptide produced greater than 90% inhibition. The concentrations of (Hyp t°°, Nlet°2)M(I)(98-110) producing 50% inhibition of

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enzyme activity (IC50 values) were 0.37 and 0.47 mM for the low and high concentrations of the protocollagen substrate used, respectively. These results indicate that the synthetic collagen peptide is recognized by and interacts with human lysyl hydroxylase, either as an inhibitor or alternate substrate. The peptide containing [3H]lysine was used as

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substrate for normal human fibroblast lysyl hydroxylase. Tritium release from (Hyp I°°, Nle 1°2, [ 3H]Lyst03 ) cd(I)(98-110) by human lysyl hydroxylase was linear over approximately a 60 rain incubation period under the conditions of the assay (Fig. 4). Blank values for the assay performed in the absence of cell extract averaged 615 dpm. These data indicate a specific enzyme-catalyzed formation of [3H]water from the peptidyl [~H]lysine residue within the synthetic peptide substrate.

TABLE II TRITIUM RELEASE FROM RADIOLABELLED SYNTHETIC PEPTIDE A N D N A T U R A L PROTOCOLLAGEN SUBSTRATES BY N O R M A L AND MUTANT H U M A N LYSYL HYDROXYLASE Lysyl hydroxylase activity (mean + S.E.) was determined by measuring formation of [ 3H]water. The normal and mutant enzymes were prepared from cultures of human dermal fibroblasts. The activities of the lysyl hydroxylase preparations from cells of the patient with Ehlers-Danlos syndrome are expressed as a percent of those from normal cells Expt.

Cell line

Substrate

Lysyl hydroxylase activity (dpm 3H released/h per mg protein)

Percent control

1

1271 3252 3839 3252

Synthetic peptide ~ Synthetic peptide " Natural protocollagen b Natural protocollagen b

1940 + 133 641 _+214 9800 + 255 2955 _+159

100 33 100 30

2

(normal) (mutant) (normal) (mutant)

" Synthetic peptide substrate was (Hyp 1°°, Nle 1°2, [3H]Lysl°3)al(I)(98-110). See text for sequence. b Protocollagen substrate was [3H]lysine-labelled, unhydroxylated protocollagen prepared as described in Materials and Methods.

150 Hydroxylation of radiolabelled synthetic peptide as well as crude preparations of radiolabelled protocollagen by extracts of normal and mutant human fibroblasts is summarized in Table lI. Fibroblasts from a patient with Ehlers-Danlos syndrome type VI had protocollagen lysyl hydroxylase activity that was only 30-33% of the activities from control cells. The specificity of the assay for lysyl hydroxylase using (Hyp m°, Nle m2, [~H]Lysl°3)M(I)(98-110) as substrate was confirmed by the fact that identical results were obtained using either this peptide or the standard preparation of [ 3H]lysine-labelled protocollagen as substrate. Discussion

The radiolabelled synthetic peptide substrate was choosen to correspond closely to amino acid residues 98 to 110 in the M(I) chain of chick (as well as rat and calf) collagen [22]. Lysine-103 from most species is always extensively hydroxylated [22,41], and this hydroxylysine is a major site of carbohydrate attachment [41,42]. The corresponding positions in the a2 chain of type I collagen as well as in the M(III) chains are also hydroxylysine [43 45]. (Hyp)°°, Nle 1°2, [3H]Lysl°~)M(I)(98 110) was synthesized to contain hydroxyproline rather than proline in position 100 so that it would not be a substrate for protocollagen prolyl hydroxylase. The use of hydroxyproline rather than proline probably slightly decreased the affinity of lysyl hydroxylase for the peptide but also prevented apparent inhibition of lysyl hydroxylase activity by prolyl hydroxylase in the tissue extract competing for the same substrate [46]. Methionine-102 in the cd(I) sequence was replaced with the isosteric norleucine residue [47,48] to avoid potential problems in peptide synthesis presented by a sulfurcontaining amino acid [28]. The most direct approach to the synthesis of a [ 3 H]lysine-containing collagen peptide, the protection and incorporation of [3H]lysine into the peptide, was attempted. This de novo synthesis of a peptide with a radiolabelled amino acid is similar to the approach originally used by Du Vigneaud et al. [49] but is complicated by the requirement for differential protection of two functional groups in the amino acid. A rather modest amount of

[3H]lysine was used as starting material, and the synthesis of the peptide was undertaken on a small scale to assess the usefulness of the approach. The desired radiolabelled synthetic peptide, (Hyp 1°°, Nle m2, [3H]Lysl°3)cd(I)(98-110), was obtained at a specific activity of 5.8 mCi/mmol. The following considerations probably explain the low recovery of tritium in the final product, which was only 1.7% of the starting material. The small scale of synthesis of the [3H]lysine derivatives (0.25 mmol) produced lower recoveries than would have been obtained with a larger scale procedure. Less than an equimolar amount of N%Boc-N'-o-chloro-Z[3H]lysine was available for coupling to the peptidyl-resin, thus this reaction was not driven to completion. Also, a significant number of the peptide chains were prematurely terminated after the coupling of N%Boc-NF-o-chloro-Z-[3H]lysine, leading to the production of the peptide ([3H]Lysm3)cd(I)(103-110) and decreasing the recovery and specific activity of the desired product. The cause of this termination reaction was not investigated. The large loss of tritium sustained in converting the peptidyl-resin to the purified peptide (other than the removal of tritiated impurities) was probably due to hydrogen-tritium exchange that occurred during the catalytic hydrogenation used to deprotect the NG-nitroarginyl residue. Use of anhydrous HF for simultaneous cleavage and deprotection of the peptide [50] would be preferred. However, the present approach of incorporating a [3H]lysine derivative suitable for solid-phase peptide synthesis was successful in so much as the desired product was a substrate in a tritium-release assay for protocollagen lysyl hydroxylase. (Hyp 1°°, Nle u)2, [3H]Lysl°3)M(I)(98-110) is similar in size and sequence to the non-radiolabelled synthetic peptide termed L-I (Ala-ArgGly-Ile-Lys-Gly-Ile-Arg-Gly-Phe-Ser-Gly) shown to be a substrate for lysyl hydroxylase by Kivirikko et al. [6]. The L-I peptide has been used extensively in an assay measuring the stoichiometric release of )4CO 2 from the cosubstrate c~keto[14C]glutarate [6]. The interaction of (Hyp 1°°, Nlem2)M(I)(98 110) with lysyl hydroxylase was almost identical to that of L-I. The ICs0 values for inhibition of the normal human lysyl hydroxylase (Fig. 3) were in good agreement with the reported

151 K m values of either the chick embryo [6,9] or human placenta [4] enzymes for L-I. These results were the basis for using the radiolabelled peptide at a final concentration of 0.5 mM in the assay reported here. At this K m concentration of (Hyp ~°°, Nle 1°2, [3H]Lysl°3)al(I)(98-110), the velocity value of the normal lysyl hydroxylase (1940 dpm 3H released per h per mg protein; Table II) corresponds to 0.6 nmol peptidyllysine hydroxylated/h per mg protein (if there is no predominant isotope effect). Assuming that the enzyme has similar ]':cat values with both (Hyp l°°, Nle 1°2, [3H]Lysl~3)M(I)(98-110) and the L-1 peptide and using the above velocity and the molar activity value of 3 s -1 determined with the L-I peptide [4,8], the crude supernatant fraction from normal human fibroblasts used in this study contained (1-2). 10-5 mg of lysyl hydroxylase per mg of total protein. This corresponds to a 50000-100000-fold enrichment required for purification to homogeneity, a value which is in close agreement with the fold purification reported for the enzyme from the supernatant fraction of human placenta [4]. The [3H]lysine.containing synthetic peptide, therefore, is a useful substrate for the assay of human lysyl hydroxylase activity in relatively crude tissue extracts. Synthetic peptides have several advantages over preparations of radiolabelled protocollagen as substrates for lysyl hydroxylase [18]. Radiolabelled protocollagen preparations are usually not pure and, therefore, are not well-defined as to specific radioactivity of the protocollagen molecules contained within them. Assays with separate protocollagen preparations usually result in different specific enzyme activities that cannot be directly compared without extensive use of standard curves [13]. Because the radiolabelled synthetic peptide described in this report is chemically pure, relatively stable, and of a well-defined specific radioactivity, it will be useful in standardization of assays so that data obtained from different experiments and laboratories can be more readily compared. Additionally, [3H]lysine-containing synthetic peptides used in a tritium-release assay should allow greater specificity for the measurement of lysyl hydroxylase in impure tissue extracts than do non-radiolabelled peptides used in an assay of ~4C02 release from a-keto[~4C]glutarate.

This is because crude extracts contain other enzymes able to decarboxylate a-ketoglutarate [18]. Disadvantages of tritiated peptide substrates include a potentially greater susceptibility to proteolytic degradation than that of protocollagen and a slow exchange of tritium to [3H]water occurring on prolonged storage. Inclusion of proteinase inhibitors in the assay and lyophilization of the radiolabelled peptide prior to use should circumvent these problems. A [3H]lysine-containing collagen peptide of much greater specific radioactivity than that obtained by the present approach is desirable to make the assay more sensitive. The synthesis of such a high specific activity peptide on a large scale would be required for its use as a substrate in routine assays. Additionally, a longer peptide, possibly corresponding exactly to the amino acid sequence around lysine-103 in human al(I) collagen, would be expected to have a lower g m value [6] and could be used at a lower concentration. The problems encountered in the present radiolabelled peptide synthesis could be circumvented by the use of a recently introduced alternative approach to the synthesis of [3H]-lysine-containing peptides [51,52]. We are currently synthesizing such an improved substrate for protocollagen lysyl hydroxylase, using tritium gas for the catalytic reduction of a collagen peptide containing an acetylenic analog of lysine [52] to yield a high specific activity [4,5-3H]lysyl residue. (Hyp l°°, Nle 1°2, [3H]Lysl°3)al(I)(98-110) or improved radiolabelled peptide substrates may help resolve properties of the genetically altered lysyl hydroxylase by defining the affected part of the catalytic reaction that results in reduced enzyme activity.

Acknowledgements We are grateful to Gwin S. Kellum for skillful technical assistance. This research was supported by NIH Grant GM28144 (D.B.G.) and Grant 1062-31 from the Woodruff Research Fund of Emory University (P.P.D.).

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