THE JOURNAL OF BIO~GICALCHEMISTRY Vol. 243, No. 9, Issue of May 10, pp. 2390-2398, 1968

Printed in U.S.A.

A Comparison of Glycopeptides Derived from Soluble and Insoluble Collagens*

(Received for publication, August 28, 1967)

LEON W. CUNNINGHAM AILD JOHN D. FORD

From the Department of Biochemistry, Vanderbilt University School qf Medicine, Nashville, Tennessee 36203

SUMMARY

Digestion of preparations of insoluble collagen of guinea

fraction, “soluble collagen,” decreased with the age of the animal (2) have led to a considerable interest in the chemical basis of this “aging” phenomenon. The concept of the progressive formation, extracellularly, of covalent cross-links in collagen fibrils has recently received direct support from the reports of Bornstein, Kang, and Piez (3) and of Bornstein and Piez (4) and the isolation of peptides in which covalent bonds between collagen o( chains have been found to result from the oxidative deamination of lysine residues in the NHz-terminal region and a subsequent aldol type condensation between residues in adjacent chains. It is by no means certain, however, that this bond constitutes the only interchain cross-link in collagen. One of the earliest postulates concerning cross-linking mechanisms was that of Grassman and Kuhn (5), who pointed to the ubiquitous presence of carbohydrate in collagen and to the dramatic solu- bilizing effect of periodate upon insoluble collagen. Nishihara (6) has reported a similar increase in solubility following treat- ment of insoluble collagens with oc-amylase, and Oneson and Zacharias (7) have reported a direct correlation between the viscosity and the carbohydrate content of dispersions of tendon collagen. These and relat,ed studies have been incorporated by Hormann (8, 9) and by Gallop, Seifter, and Meilman (10) into a hypothetical cross-link in which a carbohydrate residue is involved in glycosidic linkage, ester linkage, or both to the collagen chains. More recently, Ulumenfeld et al. (11) have presented evidence which they interpreted as indicating that glucose and galactose are present only as monosaccharides linked glycosidically to the protein and that the only available site for ester linkage was C-6 of glucose.

pig skin with collagenase and trypsin led to the production of two distinct glycopeptide fractions. The lower molecular weight fraction was similar in amount and in composition to the single predominant glycopeptide fraction obtained by similar treatment of soluble collagen. The major component in this fraction had the same chemical and physical charac- teristics as the glycohexapeptide, Gly-Met-Hyl(-Gal-Glc)- Gly-His-Arg, which had been characterized previously in similar digests of soluble collagen.

The second and higher molecular weight glycopeptide frac- tion (which contained no hydroxylysine) was found only in insoluble collagen. The major amino acids and carbo- hydrates present in this fraction were aspartic and glutamic acids, glycine, alanine, serine, proline, glucose, galactose, mannose, glucosamine, galactosamine, and sialic acid. The fraction appeared to be quite heterogeneous, and it has not yet been possible to establish whether interpeptide cross- links were present. This fraction was, however, increased in samples of perchlorate-insoluble collagen identical with those in which Hormann reported evidence for covalent cross-links involving carbohydrate.

Alkaline hydrolysis of soluble or insoluble collagen led to the isolation of closely similar quantities of both O-Hyl- (-Gal-Glc) and 0-Hyl(-Gal), thus establishing the presence of limited numbers of monosaccharide side chains in both forms of collagen. These analyses, as well as studies of partial acid hydrolysates of purified hydroxylysine-containing glycopeptides, showed the presence in mammalian skin collagen of a disaccharide in which C-l of D-glucose is linked to D-galactose, and C-l of D-galactose to the B-hydroxyl group of hydroxylysine. No evidence was found for the participation of this carbohydrate prosthetic group in cross- linking related to the conversion of soluble collagen to in- soluble collagen.

The discoveries that only a portion of the collagen of connective tissue could be extracted by dilut,e salt or acid (1) and that this

* This workwas supported by Grant GB-5994 from the National Science Foundation and by a grant from the Middle Tennessee Heart Association.

Our recent demonstration (12, 13) that a large portion of the carbohydrate in soluble collagen was linked as a disaccharide 0-glycosidically to a hydroxylysine residue led us to undertake similar studies of insoluble collagen in order to permit a direct comparison and evaluation of the participation of this carbo- hydrate in interchain linkages.

EXPERIMENTAL PROCEDURE

Xaterials

Citrate-soluble collagen was prepared by the method of Gallop and Seifter (14) from the skins of guinea pigs weighing between 250 and 400 g. Insoluble collagen was obtained from similar guinea pig skins by a slight modification of the method of Veis, Anesey, and Cohen (15). In brief, this procedure involved

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Issue of May 10, 1968 L. W. Cunningham and J. D. Ford 2391

the repeated washing of l-square inch sections of shaved guinea pig skins, first with water and then with 10% sodium chloride at 0” for at least 18 hours. The sections were next dehydrated with cold acetone and extracted with ether. The tissue was then extracted sequentially in the cold with 0.15 M NatHPO+, pH 8.0, water, phosphate-citrate buffer, pH 3.5 (718 ml of 0.1 M citric acid plus 282 ml of 0.2 M Na2HP04), water, and 2% acetic acid. The swollen skins were freed of the hair layer, and the residue was again extracted with pH 3.5 phosphate-citrate buffer and pH 8.0 phosphate buffer. The final residue of “insoluble col- lagen” was washed exhaustively wibh cold water, lyophilized, and ground into a fine powder.

A preparation of highly cross-linked collagen was obtained by the repeated extraction of samples of insoluble collagen with 2 br sodium perchlorate as described by Hormann (9). After four extractions, approximately 25% of the original material re- mained. This residue was dialyzed exhaustively against distilled water, lyophilized, and stored as “perchlorate-insoluble collagen.”

Collagenase was prepared from commercial preparations of crude Clostridium histolyticum collagenase by a modification (13) of the gel filtration procedure of Keller and Mandl (16).

Trypsin, crude collagenase, Galactostat, and Glucostat were obtained from Worthington. Pronase was purchased from Calbiochem. Orcinol was bt o ained from Matheson Coleman and Bell (Division of the Matheson Company, Inc., East Ruther- ford, New Jersey), ninhydrin and DL( +)-allo- hydroxylysine hydrochloride from Mann, carboxymethyl cellulose from Gallard- Schlesinger Chemical Manufacturing Corporation, New York, and Sephadex from Pharmacia.

Methods

Carbohydrate Analyses-The hexose content of column effluents was routinely analyzed by the automated orcinol procedure (17) with galactose as standard. Insoluble materials were analyzed for hexose manually by a slight modification of the method of Weimer and Moshin (18).

Sialic acid was determined by the method of Svennerholm (19) as modified by Miettinen and Takki-Luukkainen (20).

The method of Park and Johnson (21) was used for the analysis of reducing sugar.

Carbohydrate-containing materials were detected on paper chromatograms with the aniline-phthalate spray described by Partridge (22). Glucosamine and galactosamine were deter- mined on g-hour hydrolysates (4 NHC~, IlOo) with the use of the model 120 Beckman-Spinco automatic amino acid analyzer.

;Imino Acid and Peptide Analyses-The presence of peptides in column effluents was detected and estimated semiquantita- tively by the ninhydrin procedure of Moore and Stein (23) or by the measurement of absorbance at 285 mp. Quantitative amino acid analyses (24) were obtained with the model 120 Beckman- Spinco automatic amino acid analyzer. Acid hydrolysis was carried out in 6 N HCl at 110” for 20 or 70 hours.

Preparation of Glycopeptides by ProteolysisInsoluble or perchlorate-insoluble collagen (1.5 g) was suspended in 20 ml of 0.05 M Tris-HCl, pH 7.4, containing 0.01 M CaCl2. Digestion was begun by the addition of 15 mg of purified collagenase, and this mixture was stirred at 40” for 24 hours. During the first 3 hours the pH was maintained at 7.4 by the automatic addition of 2 N NaOH with the aid of a Radiometer titrigraph; the re- mainder of the digestion was allowed to proceed in a closed

system under toluene vapor. After 24 hours the pH was raised to 8.0 and 30 mg of trypsin were added. The pH was main- tained at 8.0 automatically for 3 hours; the remainder of the trypsin digestion was carried out in a closed system as before.

Following this 2-day digestion period, the solution was brought to pH 5.0 and insoluble material, constituting about 10% of the original weight of insoluble collagen, was removed by centrifuga- tion. In the case of perchlorate-insoluble collagen, approxi- mately 15% of the original material remained insoluble.

Fractionation of Glycopeptides by Column Chromatography-A 12-ml sample of the soluble fraction from the collagenase-trypsin digest of insoluble or perchlorate-insoluble collagen was subjected to gel filtration on a Sephadex G-25 column (bed volume, 700 ml; void volume, 240 ml) which was equilibrated and eluted with 0.1 M pyridine-acetate buffer, pH 5.0. The effluent was collected in IO-ml fractions at the rate of 72 ml per hour. Three carbohydrate-containing fractions were obtained; they were designated POOLS I, 11, and III (Fig. 1, below). These fraction were lyophilized and stored at 0”.

Further fractionation of Pool II was carried out on a column, 1.8 x 18 cm, of carboxymethyl cellulose which had been equil- ibrated with 0.01 M citrate buffer, pH 3.2, containing 0.005 M

NaCl. Elution was carried out at the rate of 45 ml per hour with this same buffer until 30 ml had been collected; a linear gradient to 0.1 M citrate-O.05 M NaCl, pH 3.2, was then begun (170 ml of each buffer), 3-ml fractions being collected. From this fractionation of Pool II, three carbohydrate-containing fractions were obtained. They were lyophilized and stored at 0” as Peaks 1, 2, and S (Fig. 2, below).

Approximately one-third of Pool I was dissolved in 1 ml of 0.1 M pyridine-acetate, pH 5.0, and subjected to gel filtration on Sephadex G-100 (bed volume, 65 ml; void volume, 19 ml) in the same buffer at a flow rate of 6 ml per hour. Resolution of approximately four hexose-containing fractions, A, B, C, and D, was observed (Fig. 3, below). The optical density of each tube was also measured at 285 rnp as a simple but very approximate index of the presence of other components (in the presence of pyridine).

Pool 111 contained only about 10 % of the total hexose of the soluble material obtained after proteolytic digestion, and has not been investigated further.

Alkaline Hydrolysis of Glycopeptides from Soluble and Insoluble Collagens-Soluble collagen (1.5 g) from guinea pig skin was suspended in 20 ml of 0.05 M Tris-0.01 M CaCl%, pH 7.4, and gelatinized by heating at 60” for 25 min. The solution was cooled to 40” and digestion with collagenase (only) was carried out exactly as described above. The digest was fractionated by gel filtration on Sephadex G-25 (bed volume, 700 ml) with 0.1 M

pyridine-acetate, pH 5.0, as eluent (Fig. 5, below), The glycopeptide fraction was pooled, dried by lyophilization,

and dissolved in 10 ml of 2 N NaOH in a stoppered Vycor test tube. Hydrolysis was then carried out for 16 hours at 90” and the resulting hydrolysate was fractionated (13) on a column of Sephadex G-25 (bed volume, 700 ml) which was equilibrated and eluted with 0.1 M pyridine-acetate, pH 5.0. The glycoside- containing fraction was pooled, lyophilized, and dissolved in about 3 ml of 0.2 N sodium citrate buffer, pH 3.25. A 0.5-ml aliquot was applied to a column, 159 x 0.9 cm, of Dowex 50 (Technicon Chromobeads, Type A, Lot 129A) which had been equilibrated with the same buffer at 55”. Elution (68 ml per hour) was then carried out stepwise with a change from this

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TABLE I Composition of residue fraction after collagenase-trypsin

digestion of insoluble collagen

Component Analysis

Amino acida Hydroxyproline .................. Aspartic acid. ............... ..... Threonine ........................... Serine ........... .................... Proline ................................ Glutamic acid ................... Glycine. .......................... Alanine ...... .............. ...... Valine ......................... .... Methionine. ........................... Isoleucine. ....... ....... ....... Leucine .............. ................. Tyrosine ................. ... ..... Phenylalanine ........................ Hydroxylysine ........................ Lysine .................................. Histidine. ............ .................. Arginine ................. ... Half-cystine ................. ......

Carbohydrateb Hexose .............................

21 42 42 65 73 89

219 89 50 20 29

GO 31 33

6 30 10 35 48

1.3

-

a Expressed as residues per 1000 residues in the 20-hour hydroly- sate. TABLE II

b Expressed as percentage. Analyses of soluble and insoluble collagens from guinea pig skin

buffer to 0.2 N sodium citrate, pH 4.25, at 340 ml. The effluent solution was divided and part (lOyO) was analyzed directly by the ninhydrin procedure (23), while the rest was collected in l.O-ml fractions and analyzed for sugar by the automated orcinol procedure (17). Five significant hexose-containing peaks, labeled A, B, C, D, and E, were detected (Fig. 6.4, below). Each of these fractions was pooled and further purified by gel filtration on Sephadex G-10 (bed volume, 200 ml) in 0.1 M pyridine-acetate, pH 5.0. The fractions were then analyzed for sugar and amino acid contents.

A sample of insoluble collagen was treated and analyzed in the same manner, with some minor procedural differences. The gelation step prior to collagenase digestion was omitted. Follow- ing digestion, approximately 10% of the original weight of the insoluble preparation remained insoluble and was removed by centrifugation. The soluble fraction was subjected to gel filtration on Sephadex G-25, and the higher molecular weight glycopeptide material was pooled separately instead of pooling the entire glycopeptide fraction. After alkaline hydrolysis the two hexose-containing fractions were desalted on Sephadex G-10 and chromatographed on Dowex 50 as described above. Carbo- hydrate-containing Fractions A to E (Fig. 6B, below) were obtained from the lower molecular weight portion of the digest of insoluble collagen; no amino acid glycosides were obtained from the higher molecular weight fraction.

Acid Hydrolysis of Purified Collagen Glycopeptides-The Peak S glycopeptide from carboxymethyl cellulose chromatog- raphy and the Peak C hydroxylysine disaccharide from Dowex 50 chromatography following alkaline hydrolysis were each dissolved in 2 N HCI (0.01 mg of hexose per ml) and heated in an

oil bath at 105”. At increasing time intervals aliquots were removed and analyzed (21) for reducing sugar (Fig. 7, below).

RESULTS

Glycopeptides Obtained by Proteolysis of Insoluble Collagen- Sequential digestion of soluble collagen with collagenase and trypsin has previously been shown (12, 13) to yield a well defined group of relatively low molecular weight glycopeptides, all of which contain hydroxylysine. The major peptide has been studied in detail and has been shown to have the structure Gly-Met-Hyl(-Gal-Glc)-Gly-His-Arg. The structures of the minor peptides have not yet been determined, but their chromatographic behavior and composition strongly suggest that they arise from the same or very similar hydroxylysine- containing portions of the peptide chain of collagen. In order to make a direct comparison of these glycopeptides with those from insoluble collagen, the same sequential collagenase-trypsin digestion procedure was used with insoluble collagen from guinea pig skin as substrate.

Approximately lOY0 of the insoluble collagen preparation was not solubilized after digestion wit,h collagenase and trypsin. A typical analysis of the composition of this precipitate is shown in Table I. Though similar in some respects to analyses of both soluble and insoluble collagens, it differed markedly in others. It accounted at least in part for the small compositional differences (Table II) between soluble and insoluble collagen

Soluble Insoluble collagen~ collagena

Amino acidc Hydroxyproline ........ 9G 97 108 Aspartic acid ........... 46 47 53 Threonine. ........ .... 20 18 18 Serine .................. 38 40 38 Proline. ............ 125 110 124 Glutamic acid ......... 75 75 75 Glycine. ............ 321 310 297 Alanine. ............. 112 104 98 Valine. ................. 21 25 21 Methionine. ............ G.6 8.3 8.0 Isoleucine. ............. II 13 14 Leucine ................ 24 27 30 Tyrosine ............. 1.6 6.0 6.7 Phenylalanine. ......... 11 14 14 Hydroxylysine. ......... 5.5 6.0 4.5 Lysine ................. 31 34 31 Histidine. ........... 6.0 7.2 6.7 Arginine. ............... 51 55 51 Half-cystine. ........... Absent 0.9 2.6

Carbohydratea Hexose ................. 0.45-0.50 0.7-0.9 0.8-0.9 Glucosamine ........... <O.Ol 0.07 0.14 Galactosamine ......... <O.Ol 0.05 0.02

Component Perchlorate-

insoluble collagenb

a Average values from duplicate 20-hour and ‘i-O-hour hydroly- sates, extrapolated to zero time.

b Average values from duplicate 20-hour hydrolysates. c Expressed as residues per 1000 residues. d Expressed as percentage.

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preparations, but not for the significant difference in carbohy- drate contents. A detailed study of this insoluble material has not been made.

The soluble portion of the digest (which corresponded to the only fraction obtained from the comparable digestion of soluble collagen) was fractionated on Sephadex G-25. A typical experi- ment is illustrated in Fig. 1. The distribution of orcinol- and ninhydrin-reactive materials was quite similar to that which we have previously reported (12, 13) for the comparable digest of soluble collagen, with one especially notable difference. The glycopeptide fraction, collected as Pool I, tubes 25 to 34, was very much larger than the similar fraction from soluble collagen (13) and accounted entirely for the difference in hexose content of the two types of collagen. Pool 11, tubes 35 to 47, corresponded exactly with the major glycopeptide fraction from soluble collagen. Pool 111, tubes 48 to 67, was collected to obtain a comp1et.e hexose recovery from the column but was small, in terms of hexose content, in both soluble and insoluble collagen digests and has not been further investigated.

Fractionation and Analysis of Low Molecular Weight Glycopep- tides (Pool II)-Inasmuch as successful subfractionation of the major glycopeptide fraction from soluble collagen had been obtained on carboxymethyl cellulose (12, 13), we first turned our

I A (15.6)

20 30 40 50 60

FRACTION NUMBER

FIG. 1. Fractionation on Sephadex G-25 of peptides produced by a sequential collagenase and trypsin digestion of insoluble col- lagen from guinea pig skin. The eluting agent was 0.1 M pyridine- acetate, pH 5.0; lo-ml fractions were collected. 0 ~- l , hexose, determined as galactose equivalents by the orcinol reaction; n---n, peptide, determined as absorbance at 570 mp by the ninhydrin reaction.

PEAK 3

0 20 30 40 50 60 70 80 90 100 110 120 130

FRACTION NUMBER

FIG. 2. Chromatography on carboxymethyl cellulose of the low molecular weight glycopeptide fraction (Pool II of Fig. 1) of in- soluble collagen with gradient elution from 0.01 M citrate-O.005 M 1”JaCl, pII 3.2, to 0.1 M citrate-O.05 M NaCl, pH 3.2, 3.ml fractions being collected. Hexose was determined as galactose eqtlivalents.

TABLE III

Composition of low molecular weight glycopeptide fraction (Pool II, Peak 3) of insoluble collagen

Amino acid Peak 3

Hydroxylysine Histidine.. Glycine. Methionine. Arginine Lysine Glutamic acid.. Hexose. Glucose. , Galactose..... ..,.

?noles/n?ole hydvoxylysint?

1.0 1.09 rt 0.01 2.93 f 0.28 0.68 f 0.14 1.23 f 0.06 0.28 f 0.08 0.24 f 0.12 2.45 0.94 0.89

Q Average values from duplicate 20.hour hydrolysates.

attention to an identical study of the comparable fraction, Pool 11, of insoluble collagen. The results of fractionation of Pool II from insoluble collagen on carboxymethyl cellulose are shown in Fig. 2. Both the location and the quantity of the three major glycopeptide peaks in Fig. 2 corresponded extremely well to those obtained from soluble collagen. This similarity

was further borne out by the analysis of the major component (Peak 3) in Table III. The amino acid and carbohydrate compositions were essentially identical with those of the same component (Peak 3) from soluble collagen (13). Furthermore, the electrophoretic mobilities of Peak 3 peptides from soluble and insoluble collagens in pyridine-acetate buffer, pH 3.6 (13), were identical. Identical behavior was also observed on paper chromatography in ethanol-water-NH40H, 60:40: 1.

As noted earlier, detailed analyses of Peaks 1 and 2 from either soluble or insoluble collagen have not yet been carried out. However, preliminary compositional studies suggested that the comparable peptides from both types of collagen were identical, and the results of these studies were consistent with the origin of such peptides from hydroxylysine-containing regions of the collagen molecule similar to that from which Peak 3 was derived.

Fractionation and Analysis of High Molecular Weight Glycopep- tides (Pool IT)-The higher molecular weight, hexose-containing fraction (Pool I) from t.he insoluble collagen digest (which was essentially absent from soluble collagen digests) was fractionated on Sephadex G-100. A typical result is seen in Fig. 3, which shows the resolution of four glycopeptide fractions, A to D, with Fractions A and C predominating. A typical analysis of these fractions is shown in Table IV. Although obviously heterogeneous, it is clear that they were all relatively rich in acidic amino acids, glycine, alanine, serine, and proline, and contained very little basic amino acid. In contrast with the peptides of Pool 11, almost no hydroxylysine was present. The presence of glucuronic acid, glucosamine, and all of the galactosa- mine in Fraction A suggested that it contained small quantities of hyaluronic acid and chondroitin sulfates which were not removed by extraction during the purification of the insoluble collagen. The presence of mannose in Fraction C at a level approximately the same as that of galactose, and the presence of glucose, glucosamine, and a variety of amino acids indicated the existence of another type of oligosaccharide prosthetic group in the insoluble collagen preparation. It is difficult to say at this

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FRACTION NUMBER

FIG. 3. Fractionation on Sephadex G-100 of the high molecular weight, glycopeptide fraction (Pool I of Fig. 1) of insoluble colla- gen. The eluent was 0.1 M pyridine-acetate, pH 5.0; l-ml fractions were collected. O--O, hexose, determined as galactose equiva- lents; 0- - -0, absorbance at 285 rnp.

TABLE IV

Analyses of high molecular weight glycopeptide fractions from Sephadex G-200 gel filtration of Pool I fraction of

I-

collagenase-,

Component

Aspartic acid. ....... Glutamic acid ........ Glycine. .............. Alanine .............. Serine ............... Proline. ............. Hydroxyproline. ..... Hydroxylysine “Hexose” b .......... Glucosamine. ........ Galactosamine ....... Arginine ............. Half-cystine. ........ Lysine ............... Histidine. ........... Methionine .......... Threonine ............ Paline ............... Isoleucine. ........... Leucine. ............ Tyrosine ............ Phenylalanine. .......

tr2 -

-

lpsin digest of insoluble collagen

Fraction

A I B I c I D

1.0 0.9 1.1 0.6 0.8 0.4

Absent Absent

18.1 4.0 8.1

Absent Absent Absent Absent

<O.l 0.5 0.2 0.3 0.4 0.1 0.2

moles/mole aspartic acid’”

1.0 1.0 1.0 1.0 1.6 1.5 1.2 2.0 2.2 1.0 0.8 0.7 0.4 0.7 0.5 0.7 1.1 0.9

Absent 0.1 0.3 Absent <O.l <O.l

2.8 1.3 0.6 0.8 0.8 <O.l

Absent Absent Absent <O.l 0.3 0.3

0.2 0.1 0.2 0.3 0.2 0.3

<O.l <O.l <O.l Absent <O.l <O.l

0.4 0.3 0.2 0.5 0.4 0.4 0.4 0.2 0.2 0.4 0.G 0.4 0.2 0.2 0.1 0.2 0.3 0.2

a Determined in 20-hour hydrolysates. b Paper chromatographic analysis after 2 N HCI hydrolysis for

135 min at 100” showed glucose and galactose to be present in all samples. All fractions contained some glucosamine, but galac- tosamine was detected only in Fraction A. In addition, Fraction A contained glucuronic acid (and the lactone), while Fraction C contained mannose. Sialic acid was detected in all fractions prior to hydrolysis.

level of purification whether this material was related to collagen or to a glycoprotein “contaminant” of the collagen preparation.

HGrmann (8, 9) has shown that successive extraction of collagen with 2 M sodium perchlorate left, as a residue, a highly cross-linked collagen. Treatment of these preparations with

periodate or with amylase and hydroxylamine led to increased solubility and, in the latter case, to the release of osazone-forming substances. HSrmann has interpreted these data as supporting the presence of a carbohydrate cross-link in this preparation of insoluble collagen. An analysis of perchlorate-insoluble collagen is included in Table II. When it was digested with collagenase and trypsin approximately 15% was left as an insoluble residue. The solubilized portion could be fractionated on Sephadex G-25 in the usual way, with the result shown in Fig. 4. The high molecular weight glycopeptide peak, corresponding to Pool I of the insoluble collagen fractionation (Fig. l), was present, in appreciably larger amount in the perchlorate-insoluble material.

The sharp restriction of sialic acid to the Pool I material is clearly seen in Fig. 4. Further fractionation of this sialic acid- containing fraction, tubes 21’ to 31, on Sephadex G-100 gave a hexose distribution pattern quite similar to that of Fig. 3. The amino acid and carbohydrate compositions of the comparable Practions A and C were similar to those reported in Table IV for these fractions from insoluble collagen, except that the level of galactosamine and glucuronic acid in Fraction A was greatly reduced.

Alkaline Hydrolysis of Glycopeptides from Soluble and Insoluble

Collagens-Butler and Cunningham (13) and Cunningham, Ford, and Segrest (25) have shown that the carbohydrate prosthetic group of the major glycopeptide (Peak S, Fig. 2) produced by collagenase-trypsin digestion of soluble collagen consists of a disaccharide of glucose and galactose linked to the d-hydroxyl group of a hydroxylysine residue. Although 0-glycosides to serine and threonine residues are readily decomposed by alkali via fl elimination, 0-glycosides to hydroxylysine would not be expected to undergo this reaction and should therefore be rela- tively stable toward alkaline hydrolysis. Such stability was in fact observed and O-Hyl(PGalLGlc) was isolated in good yield from hydrolysates of the purified peptide after exposure to 2 N NaOH at 90” for 16 hours (13,25). It was of interest, therefore, to examine the stability to alkali of all of the glycopeptide regions of intact collagen in order to obtain a better idea of what fraction of the total carbohydrate is linked to hydroxylysine.

Direct alkaline hydrolysates of collagens could not be readily analyzed by chromatography on Dowel; 50 because the relatively

I A l12.6)

20 30 40 50 60 FRACTION NUMBER

FIG. 4. Fractionation on Sephadex G-25 of peptides produced by a sequential collagenase and trypsin digestion of perchlorate- insoluble collagen from guinea pig skin. The eluent was 0.1 M pyridine-acetate, pH 5.0; lo-ml fractions were collected. O--O, hexose, determined as galact,ose equivalents; @---A, peptide, determined as absorbance at 570 rnp by the ninhydrin reaction; q -----0, sialic acid, determined by the resorcinol reac- tion.

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low percentage of carbohydrate required application of an excessively large sample to the column. A preliminary col- lagenase digestion was therefore used. Isolation of all glycopep- tides in this digest was then accomplished in high yield from Sephadex columns, and alkaline hydrolysis was performed onthis more concentrated glycopeptide fraction.

Following collagenase digestion of soluble collagen, the digest was fractionated by gel filtration on Sephadex G-25 (Fig. 5). The entire glycopeptide fraction was pooled as indicated in this figure, subjected to alkaline hydrolysis, and again fractionated on Sephadex G-25 (13). The glycoside-containing fraction was then fractionated on a Dowex 50 column into five hexose-contain- ing peaks, A to E (Fig. 6A). Each of these peaks was desalted on Sephadex G-10 before further analysis.

Upon acid hydrolysis Peak A was found to contain neither amino acids nor galactose, and was therefore presumed to represent a trace contaminant of Sephadex, or a small portion of the hexose or other orcinol-reactive degradation products of collagen which survived alkaline hydrolysis. As Peak B con- tained no identifiable carbohydrate and was also present in control runs when no sample was applied to the column, it was an artifact of the buffer system. Peaks C, D, and E, on the other hand, all contained hydroxylysine and together accounted for more than 90% of the applied hexose. Analyses of Peaks C and D are given in Table V. The very small quantities of Peak E which were available precluded detailed analysis of this component. Hydroxylysine, glucose, and galactose could be readily detected in paper chromatograms of acid hydrolysates of Peak C, but only hydroxylysine and galactose were found in hydrolysates of Peaks D and E.

A single ninhydrin-positive component was found in each case upon paper chromatography or paper electrophoresis of Peaks C, D, and E. The movement of each component relative to a hydroxylysine standard, RHy 1, in n-propyl alcohol-water- NH,OH, 60:40:1, was 0.72 for Peak C, 0.80 for Peak D, and 0.77 for Peak E. Peaks D and E had identical mobilities of 0.75, relative to a hydroxylysine standard, upon electrophoresis in pH 3.6 pyridine-acetate buffer, somewhat higher than that of Peak C, 0.62.

The behavior of the Peak C compound on paper chromatog- raphy, on Dowex 50 chromatography, and on electrophoresis at pH 3.6 was identical with that of the 0-hydroxylysine disac-

FRACTION NUMBER

FIG. 5. Fractionation on Sephadex G-25 of the peptides pro- drlced by collagenase digestion of soluble collagen from guinea pig skin. The eluent was 0.1 M pyridine-acetate, pH 5.0; lo-ml frac- tions were collected. O--O, hexose, determined as galactose eq\livalents; L?- - -a, peptide, determined as absorbance at 570 rnp by the ninhydrin reaction.

ML

B

2 ,07 & :: 5 -05 4 s p -03

: g .Ol F

0 113 224 340 453 567 680 793 907

ML

FIG. 6. Chromatography on I)owex 50 of the hexose-containing fraction resulting from alkaline hydrolysis of glycopeptides of A, soluble and R, insoluble collagen from guinea pig skin. o--o, hexose, determined as galactose equivalents; & --A, peptides or amino acids, determined as absorbance at 570 rnr by the ninhy- drin reaction. Details of the chromatographic procedure are given in the text.

TABLE V

Composition of hydroxylysine glycosides isolated from alkaline hydrolysates of soluble and insoluble

collagens of guinea pig skin

Soluble collagen Insoluble collagen Component

Peak C Peak D Peak C Peak D

moles/mole hydrozylysine

Hydroxylysinea. 1 .O 1.0 1.0 1.0 Hexose 2.05 f 0.21.24 =t 0.22.4 f 0.21.19 f 0.2 Galactose6........ 0.90 Present 1.0 0.9 Glucosec.. 1 .O Trace 1.1 Trace

a Average values from duplicate 20-hour hydrolysates. No other amino acids were present in quantities exceeding 0.05 mole per mole of hydroxylysine.

h Galactose oxidase (13). c Glucose oxidase (13).

charide produced by alkaline hydrolysis of the previously reported (12, 13) purified glycopeptide from soluble collagen. It is also important to note that alkaline hydrolysis of this purified glycohexapeptide gave rise only to the Peak C com- ponent, and not to any D or E. Thus, when present, the latter must represent monosaccharide side chains present in the intact collagen molecule. The relative amounts of Peaks D and E were dependent to some extent upon the length of alkaline hydrolysis. This, and the close similarity of all other data characterizing these components, suggests that Peaks D and E

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2396 Glycopeptdes from Collagen Vol. 243, No. 9

MINUTES

FIG. 7. Liberation of reducing sugar during the acid hydrolysis of purified collagen glycopeptides in 2 ~rj IICl at 105”. •I , Peak S, Fig. 2; l , Peak C, Fig. 6A.

represented isomers resulting from alkali-catalyzed racemization at the (Y carbon atom of the hydroxylysine moiety during hydrol- ysis. The combined quantity of Peaks D and E was in agree- ment with the quantity of 0-hydroxylysyl galactose to be expected in guinea pig skin soluble collagen on the basis of the excess of galactose in the over-all compositional analysis (12, 13, 25). The slight asymmetry of Peak C suggested that racemiza- tion of this component might also have occurred, but this fraction emerged too shortly after the buffer change for complete resolu- tion to be expected. The absence of other carbohydrate derivatives and the actual yield of all hydroxylysine glycosides indicated that at least 75% and probably all of the hexose of soluble collagen is attached to hydroxylysine residues in the polypeptide chain t.hrough an 0-glycoside involving carbon 1 of the disaccharide, glucosyl-galactose, or of the monosaccharide, galactose.

From Table I, assuming a molecular weight of 300,000 and an average amino acid residue weight of 93, it may be calculated that both soluble and insoluble collagens contain approximately 19 hydroxylysine residues and 9 hydroxylysine-linked hexose residues per molecule. Since the majority of the hexose residues are linked as disaccharides, it may be seen that fewer than one- third of the hydroxylysine residues of collagen are substituted in this way.

In order to compare the distribution of alkali-stable carbo- hydrate side chains in soluble and insoluble collagens, a pre- liminary collagenase digestion followed by alkaline hydrolysis of the glycopeptide fraction was carried out on insoluble collagen exactly as has been described for soluble collagen. When the soluble fraction obtained from collagenase digestion was frac- tionated on Sephadex G-25, the resulting separation was comparable to that of Fig. 5 except that the higher molecular weight portion of glycopeptides, fractions 20 to 30, was larger and was therefore pooled separat’ely from tubes 31 to 46. Each of these fractions was subjected to alkaline hydrolysis, desalted on Sephadex G-10, and chromatographed on Dowex 50. No glycosylat,ed amino acids were detected in significant quantities in the hydrolysate of the higher molecular weight portion. A typical chromatogram of the hydrolysate of the lower molecular weight portion is shown in Fig. 6B. Again, five hexose-containing peaks were observed. Peaks A and B were artifacts noted previously which arose from the isolation and fractionation procedure. The elution volumes of Peaks C, D, and E coincided with those previously observed for the corresponding O-Hyl- glycosides from soluble collagen. This identity was confirmed

by the analyses included in Table V, and by the observation that the paper chromatographic and electrophoretic behaviors of Peaks C, D, and E from insoluble collagen were identical with those of the corresponding peaks obtained from soluble collagen. In the case of insoluble collagen somewhat less than 50% of the total original hexose could be accounted for as hydroxylysine glycosides. The absolute amount of hydroxylysine glycosides, however, corresponded rather closely to that observed in soluble collagen hydrolysates, suggesting that the increased carbohydrate content of insoluble collagen over that of soluble collagen is due to oligosaccharide (or oligosaccharides) linked to peptide chains by alkali-labile bonds.

Acid Hydrolysis of Purified Collagen Glycopeptides-Samples of purified Peak S glycopeptide (Fig. 2 and Table III) and of the purified Peak C hydroxylysine disaccharide (Fig. 6A and Table V) were subjected to acid hydrolysis in 2 N HCl. During the 3-hour hydrolysis, aliquots were taken for analysis of reducing sugar. Representative hydrolysis curves for both derivatives are shown in Fig. 7. It may be seen that both compounds exhibited low reducing power before hydrolysis. These values were rather variable, and it seems probable that they reflected a small contamination of the sample. It appears, therefore, that the pure disaccharide chain is nonreducing. Hydrolysis occurred rapidly, so that somewhat more than 1 eq of reducingsugarper mole of hydroxylysine was liberated in 10 min. Hydrolysis continued until approximately 2.1 moles of reducing sugar per mole of hydroxylysine have been released, after 120 min.

The rates of hydrolysis for the disaccharide side chain of the Peak S glycopeptide and the hydroxylysine glycoside w-ere indistinguishable, and showed evidence of a biphasic character. This suggests that one of the glycosidic bonds is more labile than the other. This interpretation was borne out by paper chroma- t,ography of serial aliquots of the hydrolysate of Peak C in n-propyl alcohol-water-NHIOH, 60 : 40 : 1. The chromatogram, after treatment with ninhydrin, showed the presence of the pure Peak C compound at zero time. No free hydroxylysine was detectable until after 15 min, at which time the only other ninhydrin-positive component present corresponded in RF to the Pea/c D compound, hydroxylysine-galactose. After 90 min, the only ninhydrin-positive component detected was hydroxylysine. These .data together showed that the linkage between C-l of n-glucose and the n-galactose moiety is measurably more labile to acid hydrolysis than the linkage between C-l of n-galactose and the &hydroxyl group of the hydroxylysine residue. In- creased resistance to acid hydrolysis of the latter bond is to be expected on the basis of the early studies of Moggridge and Neuberger (26) on the stability of glycosides of free and N- acylated glucosamines, although the structural relationship between the protecting, positively charged amino group and the glycosidic bond is so different in the two cases as to prevent, a priori, a prediction as to the magnitude of this effect in the case of the hydroxylysine derivatives.

These studies of the acid hydrolysis of purified collagen glyco- peptides, together with the alkali stability of these derivatives, provide the following partial structure of the hydroxylysine glycoside. Since this compound is nonreducing, C-l of both glucose and galactose must be involved in glycosidic linkage. The liberation of reducing power on acid, but not on alkaline, hydrolysis confirms the participation of 0-glycosidic links be- tween the two sugars and the d-hydroxyl groups of the hydroxy- lysine. The e-amino group has previously been shown t’o be

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Issue of May 10, 1968 L. W. Cunningham and J. D. Ford

free by chromatography, electrophoresis, and susceptibility to reaction with dinitrofluorobenzene (12, 13). The detection of hydroxylysine-galactose as the only monosaccharide derivative in alkaline hydrolysates of collagen and in partial acid hydroly- sates of purified glycopeptides suggests that glucose occurs only in linkage to galactose, which in turn is linked to hydroxylysine.

DISCUSSION

The postulate that a portion of the intermolecular cross-links in insoluble collagen consists of one or more hexose residues linked covalently to a peptide chain in each molecule is based on several kinds of observations. Grassman and Kuhn (5) have shown that, even under conditions in which peptide chain splitting was minimal, acid periodate oxidation of insoluble collagen led to a greatly increased solubility. This solubilization was ascribed to the scission of intermolecular carbohydrate bridges by oxidative cleavage between one or more vicinal hydroxyl groups, followed by hydrolysis of the remaining acetal linkage. Nishihara (6) next reported that incubation of insoluble collagens with preparations of cY-amylase led to increased solu- bility, and inferred from this that enzymatic cleavage of an a-glycoside resulted in the loss of an intermolecular bridge. The interpretation of these experiments by Nishihara is, however, clouded by the probability that the amylase preparations contained small quantities of proteolytic enzymes, and thus that the observed increase in solubility was really the result of prote- olytic cleavage in the “telopeptide” region of collagen which has been so thoroughly documented by Rubin, Pfahl, Speakman, Davison, and Schmitt (27) and by Drake, Davison, Rump, and Schmitt (28). Recently, however, Hijrmann (9) has provided strong supporting evidence for a direct action of ar-amylase on cross-links in insoluble collagen. Purified cY-amylase was found to release about 70% of the carbohydrate of soluble collagen in a form which could be readily converted into free osazones. Simi- lar treatment of insoluble collagen (prepared from bovine skin as the residue after extraction with 2 M NaClOJ did not produce free osazones unless the collagen was first treated with 0.72 M

hydroxylamine. These data were interpreted to indicate that, while the carbohydrate of soluble collagen was linked via only a glycosidic bond, that of insoluble collagen was linked inter- molecularly as a bridge-one end by a glycosidic bond and the other by an ester link. Finally, Oneson and Zacharias (7) were able to disperse samples of insoluble tendon collagen in 0.3% acetic acid, and found that the viscosity of such dispersions varied inversely with the amount of bound carbohydrate, implying again that polymerization, occurring via carbohydrate cross-links, restricted swelling of the collagen fibrils.

We have shown that the chemical nature and principle site of the attachment of carbohydrate to soluble collagen is a disac- charide of glucose and galactose linked 0-glycosidically to a hydroxylysine residue in the unusual sequence

Gly-Met-Hyl (-Gal-Gk-Gly-His-Arg

which contains three amino acids, methionine, hydroxylysine, and histidine, that are present in only very minimal quantities in collagen. Other glycopeptides accounting for the remainder of the carbohydrate in soluble collagen have not been charac- terized in detail but have been identified by their reproducible position, quantity, and content of hydroxylysine upon chroma- tography on Sephadex G-25 and carboxymethyl cellulose. It was therefore with some surprise that we observed that the

chromatographic distribution of hydroxylysine-containing glyco- peptides from insoluble collagen preparations was identical, within the limits of accuracy of our isolation procedure, with that obtained earlier with soluble collagen. These data sug- gested strongly that the hydroxylysyl glycosides are not involved in intermolecular cross-links during collagen maturation, since no evidence for the existence of altered hydroxylysine-containing glycopeptide fragments in insoluble, mature collagen was found. It may be noted that, although the conditions of isolation were not severe enough that loss of ester bonds was anticipated, this possibility must be borne in mind until the precise chemical nature of additional carbohydrate to peptide bonds, if they exist, is known. More importantly, perhaps, it must be recalled that a single carbohydrate residue per molecule of collagen (mol wt 300,000) would be sufficient to create an intermolecular bridge. Thus, since there are at least 8 to 10 residues of hexose per molecule in soluble collagen and almost twice that number in insoluble collagen, it may be difficult to be absolutely certain that none of these residues participates in such a cross-link. The weight of evidence, however, clearly does not support the involve- ment of the glucosyl-galactose side chain in such structures. The function of this type of side chain thus remains unknown. The heterogeneity of this side chain, in the form of a single galactose residue rather than the disaccharide, is reminiscent of the entirely different and much more complex oligosaccharide prosthetic groups of ovalbumin (29, 30) and ribonuclease (31). It must be considered, therefore, t,hat, as in the case of these latter proteins, the observed heterogeneity may indicate that these carbohydrates are the degradation products of a more complex oligosaccharide which serves an unknown function in biosynthesis or transport (30).

The presence of a second type of carbohydrate prosthetic group in insoluble collagen has been suggested by many investi- gations (7, 32, 33), but has remained questionable because of the difficulty in obtaining, or perhaps even defining, pure insoluble collagen. This question is not resolved in the present study since the higher molecular weight and considerably more com- plex oligosaccharide-peptide obtained as Fraction C from our preparations of guinea pig skin insoluble collagen could not be unambiguously identified as being attached to a portion of the peptide chain of collagen. The very low contents of hydroxy- lysine and hydroxyproline and the relatively large amounts of leucine, isoleucine, valine, tyrosine, and phenylalanine might seem to preclude its derivation from the polypeptide chain of collagen. This composition is, however, entirely compatible with its origin from the carboxyl-terminal peptide portion of the (~-1 chain of collagen. This statement assumes that the many similarities between guinea pig skin and rat collagens extend to this region, where Butler and Piez’ have found that) the COOH- terminal peptide region has the composition, ArguHislLyssHylz- Phel Tyr, Leu( IleB Valz Alar9 Gly,, ProZ1 GlulS SerlT Thr4 Asplo 4 - Hyp&Hyp,. The relatively high content of tyrosine, phenylalanine, leucine, isoleucine, and valine is also characteristic of compositions reported by Drake et al. (28) for the telopeptide region of calf skin collagen. The possible inference that cova- lent cross-links of oligosaccharride nature are formed in or near the telopeptide region during the “maturation” of collagen fibrils is obviously of great interest and deserves continued study.

Acknowledgments-We would like to express our appreciation

1 W. T. Butler and K. A. Piez, personal communication.

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2398 Glycopeptides from Collagen Vol. 243, No. 9

to Dr. Gayle Jacobs for helpful discussions and to thank Mr. Larry Eddelman and Mr. Hector Aguilar for their technical assistance.

16.

17.

REFERENCES 18.

1. NAGEOTTE, J., Compt. Rend. Seances Sot. Biol. F&ales, 96, 172 (1927).

2. OREKHOVITCH, V. N., TOUSTANOVSI~II, A. A., OREKHOVITCH, K. D., AND PLOTNIKOVA, N. E., Biokhimiya,iS, 55 (1948).

3. BORNSTEIN. P.. KANG. A. H.. AND PIEZ. K. A.. Proc. Nat.

19. 20.

21.

Acad. Sch. U: S. A.,‘%, 417 ‘(1966). ’ ’ 4. BORNSTEIN, P., AND PIEZ, K. A., Biochemistry, 63460 (1966). 5. GRASSMAN, W., AND KUHN, K., Hoppe-Seyler’s 2. Physiol.

Chem., 301, 1 (1955). 6. NISHIHARA, T., U. S. Patent S,OS4,852 (May 15, 1962). 7. ONESON, I., AND ZACHARIAS, J., Arch. Biochem. Biophys., 89,

271 (1960). 8. H~RMANN, H., Leder, 13, 79 (1962). 9. H~RMANN, H., Proceedings of the Seventh International Con-

gress of Gerontology, 42, 159 (1966). 10. GALLOP, P. M., SEIFTER, S., AND MEILMAN, E., Nature, 183,

1659 (1959). 11. BLUMENFELD, 0. O., PAZ, M. A., GALLOP, P. M., AND SEIFTER,

S., J. Biol. Chem., 238, 3835 (1963). 12. BUTLER, W. T., AND CUNNINGHAM, L. W., J. Biol. Chem., 240,

PC3449 (1965). 13. BUTLER, W. T., AND CUNNINGHAM, L. W., J. Biol. Chem., 241,

3882 (i966). 14. GALLOP. P.. AND SEIFTER. S.. in S. P. COLOWICK AND N. 0.

KAPLAN (Editors), Methods in enzymology, Vol. VI, Aca- demic Press, New York, 1963, p. 635.

15. VEIS, A., ANESEY, J., AND COHEN, J., J. Amer. Leather Chem. Ass., 66, 548 (1960).

22. 23. 24.

25. CUNNINGHAM, L. W., FORD, J. D., AND SEGREST, J. P., J. Biol. Chem., 242, 2570 (1967).

26. MOGGRIDGE, 12. C. G., AND NEUBERGER, A., J. Chem. Sot.,

27.

28.

29.

30.

31.

32. 33.

KELLER, S., AND MANDL, I., Arch. Biochem. Biophys., 101, 81 (1963).

JUDD, J., CLOUSE, W., FORD, J., VAN EYS, J., AND CUNNING- HAM, L. W., Anal. Biochem., 4, 512 (1962).

WEIMER, H. E., AND MOSHIN, J. R., Amer. Rev. Tuberc. Pul- monary Dis., 68, 594 (1953).

SVENNERHOLM, L., Biochim. Biophys. Acta, 24, 604 (1957). MIETTINEN, T., AND TARKI-LUUKKAINEN, I. T., Acta Chem.

Stand., 13, 856 (1959). PARK, J. T., AND JOHNSON, M. J., J. Biol. Chem., 181, 149

(1949). PARTRIDGE, S. M., Nature, 164, 443 (1949). MOORE, S., AND STEIN, W. H., J. Biol. Chem., 176,367 (1948). SPACKMAN, D. H., STEIN, W. H., AND MOORE, S., Anal. Chem.,

30, 1190 (1958).

745 (1938): RUBIN, A. L., PFAHL, D., SPEAKMAN, P. T., DAVISON, P. F.,

AND SCHMITT. F. 0.. Science. 139, 37 (1963). DRAKE, M. P., ‘DAVISON, P. F:, BUMP, s., AAD SCHMITT, F. O.,

Biochemistry, 6, 301 (1966). CUNNINGHAM, L., FORD, J. D., AND RAINEY, J. M., Biochim.

Biophys. Acta, 101, 233 (1965). CUNNINGHAM, L. W., Proceedings of the Fourth International

Conference on Cystic Fibrosis, Berne and Grindelwald, 1966, Karger A. G., Basel, in press.

PLUMMER, T. II., JR., AND HIRS, C. H. W., J. Biol. Chem., 239, 2530 (1964).

SPIRO, R. G., J. Biol. Chem., 242, 1923 (1967). ROBERT, L., AND PARLEEAS, J., Bull. Sot. Chim. Biol., 47.

1853 (1965).

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Leon W. Cunningham and John D. FordA Comparison of Glycopeptides Derived from Soluble and Insoluble Collagens

1968, 243:2390-2398.J. Biol. Chem. 

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