Nonenzymatic Glucosylation of Lysyl and Hydroxylysyl Residues in Type I and Type II Collagens

Collagen ReI. Res. Vol. 4/1984, pp. 427-439

Nonenzymatic Glucosylation of Lysyl and Hydroxylysyl Residues in Type I and Type II Collagens ANDREA J. PEREJDA, EDWARD and JOUNI UlTrO

J.

ZARAGOZA, EILEEN ERIKSEN

Department of Medicine, UCLA School of Medicine, Division of Dermatology, HarborUCLA Medical Center, Torrance, CA 90509, USA.

Abstract Nonenzymatic glucosylation of type I and type II collagens was examined by incubating collagen substrates with D-glucose in vitro. In one set of experiments, unlabeled collagen was incubated with 4 C]-glucose and the incorporation of [14C]radioactivity into protein was determined by TCA precipitation. The incorporation was dependent on the concentration of glucose and the time of incubation. The glucosylated product was also examined by SDS-polyacrylamide slab gel electrophoresis. The results indicated that both a1 (1)- and a2(I)-chains of type I collagen were glucosylated and the glucosylation occurred both with native and denatured collagen as substrate. In further studies [3Hl-Iysine-Iabeled collagens were glucosylated, the products reduced by NaBH4 , and the [3Hl-Iysine-derived residues were separated by amino acid analyzer. After a 144 h incubation in vitro, 18.9 % of [3Hl-Iysyl residues and 36.5 % of [3Hl-hydroxylysyl residues in type I collagen were substituted with glucose. In contrast, 47.9 % of [3Hl-Iysyl residues and 68.1 % of [3Hl-hydroxylysyl residues in type II collagen were glucosylated after 144 h incubation. Based on quantitative amino acid analyses of the substrates, these values represent 27.6 lysine plus hydroxylysine residues substituted per triple-helical type I collagen molecule and 65.3 residues per triple-helical type II collagen molecule. Thus, type I and type II collagens display differential susceptibilities to nonenzymatic glucosylation. Finally, [3Hl-proline-labeled type I collagen was glucosylated to varying extents, and the glucosylated products were used as substrates for human polymorphonuclear leukocyte collagenase. No difference in susceptibility to this collagenase was noted, irrespective of the extent of glucosylation.

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Key Words: collagens, type I and type II; human leukocyte collagenase; nonenzymatic glucosylation. Introduction It has long been recognized that glucose can bind in ketoamine linkage to free a- and E-amino groups of proteins, both in vivo and in vitro (for review, see Bailey, 1981; Brownlee and Cerami, 1981; Bunn, 1981; Perejda and Uitto, 1982). This nonenzyma-

428

A.J. Perejda, E.]. Zaragoza, E. Eriksen and J. Vitto

tic glucosylation reaction occurs via initial formation of a Schiff base, which can be converted to a stable keto amine through an Amadori rearrangement (Higgins and Bunn, 1981). Nonenzymatic glucosylation of proteins was first demonstrated for hemoglobin A, and elevated levels of glucosylated hemoglobin Ale were noted in diabetic patients whose blood sugar levels were poorly controlled (Bookchin and Gallop, 1968; Fliickiger and Winterhalter, 1976; Gabbay et al., 1977; Gonen et al., 1977; Kennedy et al., 1979 a). Previously, glucosylation of serum albumin (Dolhofer and Wieland, 1979 a), lens crystallin (Stevens et al., 1978), insulin (Dolhofer and Wieland, 1979 b), and erythrocyte membrane proteins (Miller et al., 1980) have been demonstrated in human subjects with diabetes mellitus. More recently, type I collagen, the major interstitial collagen in connective tissues, as well as basement membrane collagen preparations, have been shown to be subject to nonenzymatic glucosylation (Rosenberg et al., 1979; Cohen et al., 1980; Uitto et al., 1982; LePape et al., 1981 a, LePape et al., 1981 b). Based upon these observations, it has been suggested (Perejda and Uitto, 1982) that nonenzymatic glucosylation of proteins may contribute to development of long-term complications of diabetes, such as basement membrane thickening, scleroderma-like skin changes, joint contractures, and alterations in vascular permeability. Some of these complications involve collagen accumulation in vascular as well as other interstitial tissues. Indeed, it has been demonstrated that the solubility of collagen in diabetic tissues is decreased, as compared to controls (Golub et al., 1978; Chang et al., 1980; Schnider and Kohn, 1981). The observations reported in this study demonstrate directly the nonenzymatic glucosylation of type I and type II collagens. Furthermore, we report the relative extent of glucosylation of lysyl and hydroxylysyl residues in the a-chains. Finally, we have tested the hypothesis that nonenzymatic glucosylation of soluble type I collagen alters its susceptibility to degradation by human polymorphonuclear leukocyte collagenase. Materials and Methods Preparation of collagen substrates

Radioactive type I and type II collagens were prepared from 17-day old chick embryo tendons and sterna, respectively, by incubation with L-[4,5- 3 H(N)]-lysine or L[2,3-3 H]-proline (New England Nuclear) in modified Krebs medium, pH 7.6, containing 2 % dialyzed fetal calf serum (GIBCO), 50 fAg/mL ~-aminopropionitrile, and 50 fAg/ ml ascorbic acid, at 37°C for 5 h (Dehm und Prockop, 1971). Following addition of PM SF (3 mM), NEM (10 mM), a, a'-dipyridyl (3 mM), cycloheximide (100 fAg/m!) and NazEDTA (20 mM), the incubation mixture was homogenized with a Polytron tissue homogenizer and centrifuged at 15,000 x g for 30 min at 4°C. The collagenous proteins in the supernatant were precipitated with 176 mg/ml ammonium sulfate. The precipitate was collected by centrifugation, resuspended in 0.5 M acetic acid, and subjected to digestion with 100 fAg/ml pepsin, first for 5 h at 25°C and then for 18 h at 4°C. The pepsin-solubilized, radioactively labeled collagen was precipitated with 10 % NaCI for 18 h at 4°C, dialyzed versus 10 mM acetic acid, and centrifuged at 26,000 x g for 3 h to obtain soluble collagen substrate. Unlabeled type I collagen was prepared from human placenta by homogenization in 0.5 M acetic acid, limited pepsin proteolysis of the acetic acid insoluble material, and

Nonenzymatic Glucosylation of Collagens

429

differential salt precipitation (Bornstein and Sage, 1980). Purity of type I collagen was determined by electrophoresis of the 2.6 M NaCl precipitate. In vitro glucosylation of type I and type II collagens

D-[ 14 C-(U)]-Glucose (uniformly labeled; 329 mCi/mmoie, New England Nuclear) was purified using the technique reported by Higgins and Bunn (1981) before use. Human placental type I collagen was glucosylated by incubation of 100 Ilg collagen at 3rC in either 20 mM sodium phosphate buffer or 50 mM Tris HCI buffer, pH 7.6, with 4 C]-glucose, 5.5 X 10 5 dprnlmmole. The total glucose concentration varied from o to 1000 mgldl. [3H]-Lysine-Iabeled type I or type II collagen (spec. activity 600 cprnlllg) or eH]proline-labeled type I collagen (spec. activity 4400 cpm/Ilg collagen) were glucosylated with 100,500 or 1000 mgldl D-glucose in the presence of 3 mM NaN J at 37°C for the time periods indicated. [3H]-Lysine-Iabeled collagen was used as substrate to determine the extent of glucose substitution at lysine and hydroxylysine residues by amino acid analyses (see below), while [3H]-proline-Iabeled type I collagen was used as substrate for collagenase digestion.

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Collagenase digestion

Partially purified collagenase from human polymorphonuclear leukocytes was the generous gift of Dr. Lasse Ryhanen (Ryhanen et aI., 1982). Collagenase was dialyzed versus 50 mM Tris-HCI, pH 7.8, containing 150 mM NaCI and 10 mM CaCI 2. The incubation mixture for collagenase digestion contained 20 III of [3H]-proline-Iabeled type I collagen substrate (approximately 2.0 X 10 5 cpm), 2 Ilg bovine serum albumin, and collagenase in a final volume of 100 Ill. Incubations were performed at either 37°C or 2rc for 30 to 60 min, and digestions were terminated by the addition of Na2EDTA to a final concentration of 40 mM. In some experiments, soluble type I carrier collagen was added, and a-chymotrypsin and trypsin were added to give final concentrations of 0.2 mg/ml each. Incubation was continued for 30 min and the reaction was terminated by adding an equal volume of 40 % TCA. Precipitates were filtered and counted as previously described (Ryhanen et aI., 1982). The percent collagen degraded was calculated as follows: cpme - cpme cpme

X

100

in which cpme refers to the counts per minute of radioactivity recovered in control samples incubated without collagenase, and cpme refers to radioactivity in samples incubated with collagenase. In other experiments, the collagen degradation was monitored by SDS-polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis

Collagenase digestion products of [3H]-proline-Iabeled type I collagen were also demonstrated by SDS-polyacrylamide slab gel electrophoresis (Laemmli, 1970). Following termination of collagenase digestion with Na2EDTA (see above), the samples were immediately heated in the presence of 2 % SDS and 0.8 M urea for 5 min at 100°C and electrophoresed on 8 % polyacrylamide gels. In other experiments, sam30 Collagen 4/6

430

A.J. Perejda, E.]. Zaragoza, E. Eriksen and J. Uitto

pies of human type I collagen, incubated with [14C]-glucose, were reduced for 30 min in the presence of NaBH 4, precipitated with 20 % TCA, resuspended in 2 % SDS and 0.8 M urea, heated for 5 min at 100°C, and electrophoresed as above. The radioactive peptides on the gels were visualized by fluorography (Bonner and Laskey, 1974), and the bands were quantitated by scanning the flu oro grams with an Ortec 4310 densitometer attached to a computerized Perkin-Elmer Sigma 1 integrator.

Amino acid analyses To determine the extent of nonenzymatic glucosylation of Iysyl and hydroxylysyl residues, [3Hl-Iysine-Iabeled type I collagen was incubated, in a final concentration of 100 !J.g/ml, in 50 mM Tris-HCI, pH 7.8, containing 500 mg/dl glucose at 37°C. After varying incubation periods, the substrate was treated with excess NaBH4 at room temperature for 30 min, then hydrolyzed in 6 N HCI 120°C for 24 h, and chromatographed on a Beckman 120B amino acid analyzer using PA-28 resin. The column was equilibrated and eluted with 0.36 M citrate buffer, pH 5.28, at 55°C. Fractions were collected at 3 min intervals to yield 2.5 ml fractions and 1.0 ml aliquots were counted for [3Hl-radioactivity in 14 ml of counting cocktail (Scintiverse II, Fisher) and 1 ml methanol using a Beckman LS 7500 liquid scintillation counter. The amino acid analyzer column was calibrated, in addition to standard amino acids, with glucosyl-Iysine and glucosyl-hydroxylysine synthesized according to Schwartz and Gray (1977).

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Fig. 1. Incorporation of D_[14C(U)]-glucose into native type I collagen as a function of time and glucose concentration. Type I collagen, 100 Ilg/ml, purified from human placenta was incubated with (5.5 x 105 dpm per mmole) 4C]-glucose at 37°C for the time periods indicated and using the final glucose concentrations shown. The protein containing the bound [14C]-radioactivity was precipitated with 20 % TCA and the radioactivity was determined, as indicated in Materials and Methods.

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Nonenzymatic Glucosylation of Collagens

431

For determination of the extent of enzymatic glycosylation of type I and type II collagens, the substrates were hydrolyzed in 2 N KOH in sealed polypropylene tubes at 110°C for 24 h. To remove KOH, the hydrolysates were precipitated with KHC0 3 and HCI0 4 as described previously (Price, 1982), then rotary-evaporated and chromatographed on the amino acid analyzer, as described above. The buffer system consisted of a 0.36 M citrate buffer gradient from pH 3.25 to pH 5.28. Results Previous studies (Rosenberg et aI., 1979; Schnider and Kohn, 1981; Buckingham et aI., 1981) have suggested that type I collagen can be nonenzymatically glucosylated in vivo. To establish that type I collagen can be nonenzymatically glucosylated in vitro, we incubated native type I collagen, purified from human placenta, with [14C]-glucose at neutral pH. The purity of type I collagen was checked by SDS-polyacrylamide gel electrophoresis; the collagen polypeptides migrated in a1(1) and a2(1) positions in an apparent 2: 1 ratio. Also, 4C]-glucose was purified prior to use, as indicated in Materials and Methods. The results demonstrated that [14C]-glucose bound to native type I collagen (Fig. 1). The amount of [14C]-glucose bound to collagen increased with increasing total glucose concentration. Also, the amount of [14C]_glucose bound to TCA precipitable collagen increased with time of incubation; however, the incorporation was not directly proportional to the time of incubation but a larger increment was noted between 38 and 48 h of incubation (Fig. 1). Examination of the glucosylated type I collagen by SDS-polyacrylamide gel electrophoresis, followed by fluorography, indicated that both a1- and a2-chains of type I collagen were labeled with [14C]-glucose (Fig. 2). The ratio of radioactivity in a1(I) to that in a2(1) chains at varying time points during incubation with 4C]-glucose, was 3.3:1, as determined by scanning the fluorographs (Figs. 2 and 3). In similar experiments, the type I collagen substrate was first denatured by heating at 100°C for 5 min and then incubated with [14C]-glucose. Examination of the fluorograms, prepared from SDS-polyacrylamide gels, indicated that the non-helical polypep-

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Fig. 2. Incorporation of D_[14C(U)]-glucose into a1- and a2-chains of native type I collagen. Type I collagen substrate was incubated with [14C]-glucose, as indicated in Fig. 1, and the achains of type I collagen were separated on 6 % SDS-polyacrylamide gels. The flu oro graphs of the radioactive polypeptides .(A) were scanned and quantitated by a computerized densitometer (B).

432

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Fig. 3. Time course of incorporation of D-[14C(U)]-glucose into a1(1)- and a2(1)-chains of type I collagen. Native type I collagen was incubated as described in Fig. 1, and the products were reduced and separated on 6 % SDS-polyacrylamide gels. Fluorographs were scanned and quantitated by a computerized densitometer.

tides, like a-chains in triple-helical conformation, bound radioactive glucose. The extent of glucosylation of denatured type I collagen was slightly higher than that of native substrate. The ratio of glucosylation based upon densitometry of al(I) and a2(I) chains in denatured versus native substrate was 1.18 and 1.26 at 24 and 72 h incubation, respectively. Previous studies have established that the E-amino group of both lysyl and hydroxylysyl residues can serve an attachment site for glucose (Tanzer et al. 1972; LePape et aI., Cohen and Wu, 1981). In further studies, we examined the extent of glucosylation of these amino acids using [3H]-lysine-labeled type I collagen as substrate. After 72 and 144 h incubation with 500 mgldl glucose, the glucosylated collagen substrate was hydrolyzed in 6 N HCl and examined by amino acid analyses. Distinct peaks of radioactivity eluted in positions corresponding to those of lysine and hydroxylysine standards, as well as those of synthetically prepared glucosyl-lysine and glucosylhydroxylysine (Fig. 4). The radioactivity in the peaks corresponding to glucosyl-lysine or glucosyl-hydroxylysine at 72 h accounted for 1.0 % and 7.5 % of the total [3H]_ lysine or [3H]-hydroxylysine radioactivity, respectively (Table I). The corresponding values at 144 h were 18.9 % and 36.5 %. Quantitative amino acid analyses of the [3H]_ lysine-labeled type I collagen used as substrate revealed lysine and hydroxylysine contents of 23.6 and 12.9 residues per 1000 amino acids (Table I). Based on the relative content of these amino acids and the percent of their glucosylation, we calculated that after 144 h reaction in vitro, the substrate contained 27.6 residues glucose per triplehelical collagen molecule consisting of three a-chains of about 1000 amino acids each (Table I). It should be noted that the relative glucosylation of hydroxylysyl residues in type I collagen was consistently greater than that of lysyl residues (Table I).

Nonenzymatic Glucosylation of Collagens

433

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Fig. 4. Separation of eHJ-lysine and [3HJ-hydroxylysine from their glucosylated derivatives by an amino acid analyzer. [3HJ-Lysine-labeled type I collagen was incubated with D-glucose in a final concentration of 500 mg/dl for 144 h at 37°C. The glucosylated substrate was reduced with NaBH4 and hydrolyzed in 6 N HCL The [3HJ-labeled amino acids were chromatographed on an amino acid analyzer column, as indicated in Materials and Methods. The elution positions of glucosylated and unsubstituted hydroxylysine and lysine residues are based on corresponding quantitative standards detected by ninhydrin reaction. Similar studies were performed to examine the extent of nonenzymatic glucosylation of [3Hl-lysine-labeled type II collagen. After 72 h incubation in the presence of 500 mgl dl D-glucose, 2.1 % of the lysyl residues and 0.7 % of the hydroxylysyl residues were glucosylated, as determined by amino acid analyses of the borohydride-reduced hydrolysates. Following 144 h incubation, the corresponding values were 47.9 % and 68.1 %, respectively (Table I). Quantitative amino acid analyses of [3Hl-lysine-labeled type II collagen demonstrated that lysine and hydroxylysine contents were 17.0 and 20.0 residues per 1000. Thus, approximately 65 glucose molecules were nonenzymatically bound to each triple-helical type II collagen molecule (Table I). It is of interest that the type II collagen substrate, for which extensive nonenzymatic glucosylation was demonstrated, also had a high hydroxylysine-O-glycoside content. Specifically, 73.0 %

434

A.J. Perejda, E.]. Zaragoza, E. Eriksen and]. Uitto

Table 1. Extend of nonenzymatic glucosylation of lysyl and hydroxylysyl residues of type I and type II collagens incubated in the presence of 500 mg/dL glucose. % glucosylatedb

Type I Collagen:

Type II Collagen:

Glucosylated residues per triple-helix 144 h 72h IncubaIncubation tion

Amino Acid

Residues/ 1000'

72h Incubation

144 h Incubation

Lysine Hydroxylysine Lysine + hydroxylysine

23.6 12.9 36.5

1.0% 7.5%

18.9% 36.5%

0.7 2.9 3.6

13.5 14.1 27.6

Lysine Hydroxylysine Lysine + hydroxylysine

17.0 20.0 37.0

2.1% 0.7%

47.9% 68.1%

1.1 0.4 1.5

24.4 40.9 65.3

• Calculated from quantitative amino acid analyses of [3H]-lysine-labeled collagen substrates. b The percentage of lysine or hydroxylysine residues which were nonenzymatically glucosylated was calculated as the ratio of cpm [3H]-glucosyl-lysine or [3H]-glucosyl-hydroxylysine to the total cpm [3H]-lysine or [3H]-hydroxylysine X 100, as determined by amino acid analyses. of the hydroxylysine residues were substituted in an O-glycosidic linkage; the corresponding value in type I collagen substrate was 32.3 %. To test the hypothesis that nonenzymatic glucosylation of collagen may alter its susceptibility to digestion by a vertebrate collagenase, we first incubated type I collagen in vitro in the presence of varying concentrations of glucose. The glucosylated collagen preparations were then used as substrates for human polymorphonuclear leukocyte collagenase (Table II and Fig. 5). The incubations were performed in an enzyme: substrate ratio which allowed us to monitor the collagen degradation in the linear portion of the reaction. In three separate experiments, the digestion of [3H]-prolinelabeled collagen, which had been glucosylated to varying extents, was not significantly different from unglucosylated collagen substrate as analyzed by two independent methods (Table II and Fig. 5). The results were the same irrespective of the incubation temperature (3rC versus 2rq. Also, the addition of corresponding amounts of free glucose into the control reaction did not appear to alter the rate of collagen degradation (Table II). Discussion In this study, we have demonstrated that type I and type II collagens can be nonenzymatically glucosylated in vitro. This observation confirms previous indications that type I collagen can be glucosylated in vivo in diabetic human tissues or in animal models of diabetes (see above). We have further characterized this reaction by demonstrating that both a1- and a2-chains of type I collagen are subject to nonenzymatic

Nonenzymatic Glucosylation of Collagens

435

Table II. Digestion of nonenzymatically glucosylated eH]-proline-labeled type I collagen by human polymorphonuclear leukocyte collagenase. Control

250

Glucose Concentration (mg/dL)a 500 1000

+

Experiment No.

+

+

Collagen Degradationb

1

11,562 (22.9)

9805 (34.6)

9714 (35.2)

7658 (49.0)

10,840 (27.7)

10,605 (29.9)

9675 (35.5)

2

12,228 (18.5)

10.930 (27.1)

11,616 (22.6)

12,784 (14.8)

12,157 (19.0)

12,160 (19.0)

13,401 (10.7)

3

12,828 (14.5)

9779 (35.0)

11,237 (25.0)

12,758 (15.0)

10,428 (30.0)

a Control: Nonglucosylated [3H]-proline-labeled type I collagen was used as substrate without added glucose; +: [3H]-Proline-labeled type I collagen used as substrate was nonenzymatically glucosylated at the glucose concentrations indicated for 144 h at 37°C prior to collagenase digestion; Same as control, except that the digestions were performed in the presence of exogenous glucose in the concentrations indicated. b The values represent the radioactivity in type I collagen substrate remaining undigested by collagenase per 1.5 X 104 cpm total [3H]-labeled substrate (mean from three parallel determinations); the percent of eH]-proline-labeled substrate digested is indicated in parentheses. glucosylation. It is of considerable interest that collagen polypeptides, both in native triple-helical conformation and in non-helical form, can be glucosylated by nonenzymatic means. This contrasts the enzymatically catalyzed glycosylation of hydroxylysyl residues to form hydroxylysine-O-glycosides; the latter reactions occur only on the nonhelical collagen polypeptides and are limited by formation of the triple-helix (Kivirikko and Myllyla, 1979). Thus, it is conceivable that the native collagen fibers in the extracellular space can be subject to nonenzymatic glucosylation and that the extent of such glucosylation may be increased in clinical hyperglycemia. Separation of glucosylated lysine and hydroxylysine residues from nonglucosylated ones by amino acid analyzer allowed us to calculate the content of glucose in ketoamine linkage per collagen molecule after varying time periods of glucosylation in vitro. The results indicated that after 144 h incubation, as many as 27 lysyl and hydroxylysyl residues were substituted in type I collagen. In type II collagen, the corresponding value was 65 residues per molecule. Since type II collagen substrate was shown to have a considerably higher degree of enzymatic glycosylation, it is possible that the presence of hydroxylysine-O-glycosides in the molecule may confer properties upon the molecule which make it a better substrate for nonenzymatic glucosylation. In general, the hydroxy lysine was more readily glucosylated than lysine. Although the basis for this observation is not clear, it is conceivable that the presence of the hydroxyl group of hydroxylysine may facilitate the attachment of glucose through enhancement of the E-amino nitrogen nucleophilicity. This could occur through hydrogen bonding of the b-hydroxyl oxygen lone pairs with a hydrogen on the a-amino group. This interac-

436

A.J. Perejda, E.]. Zaragoza, E. Eriksen and J. Uitto

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Fig. 5. SDS-polyacrylamide gel electrophoresis of collagenase digestion products of glucosylated type I collagen. [3HJ-Proline-labeled type I collagen was glucosylated in the presence of 250,500 or 1000 mgldl D-glucose for 144 h, as indicated in the text. The glucosylated collagen substrates were then digested with human polymorphonuclear leukocyte collagenase. In control samples, unglucosylated type I collagen was used as substrate. Symbols: +, samples digested with collagenase; -, corresponding substrates incubated without collagenase. The electrophoretic mobilities of a1- and a2-chains of type I collagen, as well as those of a1 A and a2A, representing the amino-terminal degradation fragments of the same polypeptides, are indicated.

tion could form a relatively stable five-membered ring and might enhance the electron density on the nitrogen, thus making it a better nucleophile. Previous biochemical and ultrastructural studies have demonstrated thickening of the capillary basement membranes with accumulation of type IV collagen in skeletal muscles and glomeruli of diabetics (Beisswenger and Spiro, 1970; Kilo et ai., 1972; Kefalides, 1974; Raskin, 1983). Also, recent studies have shown thickening and induration of dermis in young patients with poorly controlled juvenile-onset diabetes (Buckingham et ai., 1981; Rosenbloom et aI., 1981; Seibold, 1982). It seemed reasonable, therefore, to postulate that excessive glucosylation might alter the metabolism of collagen, perhaps rendering it a poor substrate for vertebrate collagenase. The results of this study demonstrate, however, that nonenzymatic glucosylation of type I collagen does not alter its susceptibility to digestion by human leukocyte collagenase. Also, the presence of free glucose in concentrations found in diabetic subjects did not affect the degradation of control collagen substrate by the enzyme. Thus, it is possible that the degradation of extracellular collagen, mediated by specific collagenases, is not altered in diabetics. These results do not exclude the possibility that the intracellular degradation of newly-synthesized pro-a chains of pro collagen is changed in the diabetic milieu (Schneir et ai., 1982). Additionally, diabetes may affect collagen metabolism by changing the rate of collagen production or by interfering with fiber formation and subsequent stabilization of the fibers by covalent cross-links (see Perejda and Ditto, 1982; Chang et ai., 1980). Finally, the accumulation of type IV collagen in basement membranes may reflect increased overall protein synthesis in the hyperglycemic milieu (Pihlajaniemi et ai., 1982).

Nonenzymatic Glucosylation of Collagens

437

Acknowledgements This study was supported by USPHS, NIH grants AM-28450, GM-28833, and AM32637, and by grants from March of Dimes, The Birth Defects Foundation and American Diabetes Association, Southern California Affiliate. During a portion of this study, Dr. Perejda was supported by the American Heart Association, Greater Los Angeles Affiliate.

Abbreviations: NEM, n-ethylmaleimide; Na2EDTA, disodium ethylenediaminetetraacetate; PMSF, phenylmethylsulfonylfluoride; TCA, trichloracetic acid; arid SDS, sodium dodecylsulfate.

References Bailey, A. J.: The nonenzymatic glycosylation of proteins. Horm. Metab. Res. (Suppl.) 11: 90-94, 1981. Beisswenger, P. ]. and Spiro, R. G.: Human glomerular basement membrane: Chemical alteration in diabetes mellitus. Science 168: 596-598, 1970. Bonner, W. M. and Laskey, R. A.: A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46: 83-92, 1974. Bookchin, R. M. and Gallop, P. M.: Structure of hemoglobin Ale- Nature of the N-terminal ~-chain blocking group. Biochem. Biophys. Res. Comm. 32: 86-93, 1968. Bornstein, P. and Sage, H.: Structurally distinct collagen types. Ann. Rev. Biochem. 49: 957-1003, 1980. Brownlee, M. and Cerami, A.: The biochemistry of the complications of diabetes mellitus. Ann. Rev. Biochem. 50: 385-432, 1981. Buckingham, B. A., Uitto, ]., Sandborg, c., Keens, T., Roe, T., Costin, G., Kaufman, F., Bernstein, B., Landing, B. and Castellano, A.: Scleroderma-like changes in insulin-dependent diabetes: Clinical and biochemical studies. Diabetes Care 7: 163-169, 1984. Bunn, H. F.: Evaluation of glycosylated hemoglobin in diabetic patients. Diabetes 30: 613-617,1981. Chang, K., Uitto, J., Rowold, E. A., Grant, G. A., Kilo, C. and Williamson,]. R.: Increased collagen cross-linkages in experimental diabetes. Reversal by ~-aminopropionitrile and D-penicillamine. Diabetes 29: 778-781, 1980. Cohen, M. P., Urdanivia, E., Surma, M. and Wu, V.-Y.: Increased glycosylation of glomerular basement membrane collagen in diabetes. Biochem. Biophys. Res. Comm. 95: 765-769, 1980. Cohen, M. P. and Wu, V.-Y.: Identification of specific amino acids in diabetic glomerular basement membrane collagen subject to nonenzymatic glucosylation in vivo. Biochem. Biophys. Res. Comm. 100: 1549-1554, 1981. Dehm, P. and Prockop, D. J.: Synthesis and extrusion of collagen by freshly-isolated cells from chick embryo tendon. Biochim. Biophys. Acta 24: 358-369, 1971. Dolhofer, R. and Wieland, O. H.: Glycosylation of serum albumin: Elevated glycosyl-albumin in diabetic patients. FEBS Lett. 103: 282-286, 1979 a. Dolhofer, R. and Wieland, O. H.: Preparation and biological properties of glycosylated insulin. FEBS Lett. 100: 133-136, 1979 b. Fliickiger, R. and Winterhalter, K. H.: In vitro synthesis of hemoglobin Ale- FEBS Lett. 71: 356-360, 1976. Gabbay, K. H., Hasty, K., Breslow, ]. L., Ellison, R. c., Bunn, H. F. and Gallop, P. M.: Glycosylated hemoglobins and long-term blood glucose control in diabetes mellitus. J. Clin. End. Metabol. 44: 859-864, 1977.

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