Vol. 254, No. 3, Issue of February 10, pp, 666-671, 1979 Printed in U.S.A.

Isolation and Characterization of a Double Chain Intermolecular Cross- linked Peptide from Insoluble Calf Bone Collagen*

(Received for publication, May 11, 1978)

Norma P. Stimler$ and Marvin L. Tanzer

From the Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06032

A double chain peptide containing the sodium boro- hydride-reduced intermolecular cross-link, hydroxyly- sinohydroxynorleucine, was isolated following sequen- tial cyanogen bromide digestion and limited alkaline hydrolysis of insoluble calf bone collagen. Amino acid composition and NHz-terminal sequence analysis indi- cated that the peptide was highly acidic and consisted of 19 amino acid residues including the cross-link. Amino acid composition and automated sequence anal- ysis of this peptide before and after cleavage of the cross-link, using periodic acid, provided the data from which the following structure was deduced.

Phe-Leu-Pro-Glu-Pro-Pro-Glu-Glu-Glu-Hyl-Ala-His

I Pro-Glu,Gly,Hyl,Asp,Gly,Pro,Glu

The sequence of the larger peptide is identical with that of residues 8’ to 19” in the COOH-terminal nonhelical region of the homologous skin collagen (~1 chain. The hydroxylysine residue located at position 17” in the (Y chain of type I collagen appears to be a predominant site for intermolecular cross-link formation. Assign- ment of the smaller peptide component within the known primary structure of the collagen molecule cur- rently cannot be made.

The elucidation of the three-dimensional arrangement of collagen molecules in connective tissue fib& is essential to the understanding of the functional role of the protein. It is well known that the collagen network is stabilized by a number of interchain cross-links whose covalent structures have been described (l-3). In addition, the primary structure of most of the type I collagen molecule has recently been reported for several species (4). Little is known, however, about the number and location of the cross-links in mature tissues. Several investigators have provided evidence that the nonhelical pep- tide extensions which occur at both ends of the (Y chain subunits may play a major role in cross-linking (5-8).

Cyanogen bromide digestion of rat tail tendon collagen (6) and of insoluble collagen from bovine skin and dentin (9) yielded an intermolecular cross-linked peptide composed of the NH2- and COOH-terminal regions of adjacent molecules.

* This investigation was supported by Grants NIH AM 17220 and NIH AM 12683 from the National Instututes of Health, Grant NSF GB 31077 from the National Science Foundation, and Grant AMA 75-773 from the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed. Present address, Department of Pathology, University of California at San Diego, La Jolla, Calif. 92093. This research was presented in partial fulfillment for the requirements for the Ph.D. degree.

Several smaller double chain peptides, originating from inter- molecular cross-link formation at the same site, have been isolated from reconstituted calf skin collagen digested sequen- tially with bacterial collagenase, trypsin, and papain (10) or from insoluble calf skin collagen digested with trypsin alone (11). This cross-link occurs between a lysine or hydroxylysine residue at position gN ’ and the hydroxylysine at position 927 near the COOH terminus (12).

A second cross-linking site has been reported to occur between hydroxylysine residues at positions 87 and 17” in adjacent al chains in insoluble collagen from calf and chicken bone (7, 8). In addition, several other double chain peptides containing intermolecular cross-links have been isolated which did not arise from cross-link formation at either of these sites and have not as yet been assigned to specific loci within the collagen matrix (13, 14).

The present report describes the isolation and characteri- zation of a double chain cross-linked peptide arising from a new site of intermolecular cross-link formation involving the COOH-terminal nonhelical region of the al chain. This cross- linking site can be used in conjunction with the location of other known cross-linking sites to describe an accurate three- dimensional structure of the collagen fibril.

EXPERIMENTAL PROCEDURES

Preparation of Collagen-Insoluble calf bone collagen was pre- pared as described previously ( 15).

Reduction and Labeling of Cross-&&s-Insoluble calf bone col- lagen (10 g) was hydrated in potassium phosphate buffer, 0.05 M, pH 7.6 (25 ml/g of collagen) by stirring for 18 h at 4°C. The suspension was warmed to 37°C and 300 mg of [‘H]NaBH, (25 mg of [“H]NaBH, at 6.4 mCi/mg, New England Nuclear, mixed with 275 mg of unlabeled NaBH4, Alpha) dissolved in 5 ml of 0.01 N NaOH was added dropwise over a period of 15 min. Incubation was continued at 37°C for 1 h with occasional stirring. Excess reagent was removed by exhaustive dialysis uersus deionized water, and the collagen was lyophilized.

Preparation of Cyanogen Bromide Peptides-The “H-labeled in- soluble calf bone collagen was digested with cyanogen bromide (l:l, w/w; Eastman) in 70% formic acid (50 ml/g of collagen) at 22°C for 90 min. The peptide material was then lyophilized and fractionated by hydroxyapatite column chromatography as previously described (15). The digest was dissolved in column buffer, 0.001 M NalHPO,, pH 6.8, containing 1 M urea and 0.15 M NaCl, and adsorbed onto a column (5 x 58 cm) of hydroxyapatite (Hypatite C, Clarkson Chemical Co.) equilibrated in the same buffer. The column was eluted at 22°C with a linear gradient of 1.5 liters each of column buffer and 0.08 M NaaHI’OI, pH 6.8, containing 1 M urea and 0.15 M NaCl. After 2.5 liters of buffer had passed through the column, the gradient was converted to exponential form by the addition of 1.5 liters of 0.2 M

Na2HP04, pH 6.8, containing 1 M urea and 0.15 M NaCl to the gradient vessel from a separatory funnel with the system closed to atmospheric pressure. The flow rate was maintained at 40 ml/h, and 11-ml fractions

’ In the nomenclature used, amino acids in the NHa and COOH- terminal nonhelical regions are numbered lN to 16” and 1” to 25“, respectively. Amino acid residues in the helical region are numbered sequentially from NHa to COOH terminus.

666

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A Cross-linked Peptide from Calf Bone Collagen

were collected throughout the chromatography. Fractions were as- sayed for protein by measuring the absorbance at 230 nm and for radioactivity by scintillation counting. Peak areas were pooled as indicated in Fig. 1, desalted by dialysis versus deionized water, and lyophilized.

Limited Alkaline Hydrolysis-Cyanogen bromide peptide material eluting in the second peak from hydroxyapatite chromatography (3.5 g) was dissolved in 200 ml of 1 N KOH and heated under nitrogen in a Nalgene screw cap bottle at 1lO’C for 16 h. The digest was neutralized by the addition of HCl and lyophilized.

Peptide Purification-The peptide mixture thus obtained was divided into three portions and fractionated on column (2 x 100 cm) of Sephadex G-25 (Pharmacia) equilibrated at 22°C in 0.1 M ammo- nium bicarbonate. The column was eluted with the same buffer at a flow rate of 135 ml/h. Fractions of 4.5 ml were collected and assayed for radioactivity. Peak areas were pooled as indicated in Fig. 2, and the corresponding fractions from each chromatogram were combined for subsequent purification.

For cation exchange chromatography, Aminex A-5 resin (Bio-Rad Laboratories) was precycled (16) and equilibrated in 0.2 M pyridine acetate, pH 3.5. A column (2.5 x 60 cm) was packed at 40°C and equilibrated by washing the resin with several column volumes of equilibration buffer. Peptide samples were dissolved in 5 ml of distilled water and adjusted to pH 2 with HCl. This solution was applied to the column under nitrogen pressure (20 p.s.i.) and washed into the resin with 5 ml of equilibration buffer. The column was developed at 50°C with a linear gradient of 800 ml each of 0.2 M pyridine acetate, pH 3.5, and 0.8 M pyridine acetate, pH 5.2, at a flow rate of 100 ml/h maintained with a constant volume pump. Upon completion of the gradient the column was stripped with 8 M pyridine. Fractions of 10 ml were collected and assayed for the tritium label. Peak areas were pooled as indicated in Fig. 3A and lyophilized.

DEAE-Sephadex A-25 (Pharmacia) chromatography was con- ducted using a column (1 X 23 cm) equilibrated at 22°C in 0.1 M Tris buffer, pH 8.3. Peptide samples were dissolved in the equilibration buffer prior to application to the column and eluted with a linear gradient of 500 ml total volume from 0 to 0.4 M NaCl in 0.1 M Tris, pH 8.3. Fractions of 2.5 ml were collected at a flow rate of 35 ml/h and assayed for protein by measuring the absorbance at 230 nm and radioactivity by scintillation counting. Peak areas were pooled as indicated in Fig. 3B.

Bio-Gel P-10 (200 to 400 mesh, Bio-Rad Laboratories) chromatog- raphy was performed using a column (1 X 130 cm) equilibrated in 30% acetic acid. Lyophilized peptide material was dissolved and applied to the column in a small volume of 30% acetic acid. The column was developed at a flow rate of 6 ml/h. Fractions of 1 ml were collected and aliquots withdrawn for assay by scintillation counting and by reaction with ninhydrin following hydrolysis (17).

Chromatography on Bio-Gel P-30 (200 to 400 mesh, Bio-Rad

FRACTION NUMBER

FIG. 1. Hydroxyapatite chromatography of 10 g of CNBr digested, [“H]NaBH,-reduced insoluble calf bone collagen. A column (5 x 58 cm) was equilibrated in 1 mM Na2HPOr, pH 6.8, containing 1 M urea and 0.15 M NaCl and developed with an exponential gradient from 1 to 200 mM Na2HP0, over 4 liters at a flow rate of 40 ml/h. Fractions of 11 ml were collected and assayed for absorbance at 230 nm (-a-) and for ‘H (----). Brackets indicate pooled fractions.

667

OO- 100 It FRACTION NUMBER

FIG. 2. Sephadex G-25 chromatography (2 x 100 cm column) of 3.5 g of peptide material obtained from hydroxyapatite Fraction 2 (Fig. 1) and subjected to limited alkaline hydrolysis. The column was equilibrated in 0.1 M NHAHCO:, and eluted at 135 ml/h, collecting 4.5- ml fractions. Pooled fractions are indicated by brackets.

Laboratories) was performed using a column (0.8 X 100 cm) equili- brated in 30% acetic acid. The column was eluted under hydrostatic pressure at a flow rate of 2 ml/h. Fractions of 0.5 ml were collected and assayed as described above.

Amino Acid and Cross-link Analysis-Aliquots of the peptide samples were hydrolyzed in 6 N HCl under nitrogen for 24 h at 1lO”C. Following removal of excess HCl by evaporation, the samples were dissolved in an appropriate volume of pH 2.12 buffer and applied to the long column of a Beckman model 119 amino acid analyzer. All amino acids and cross-linking compounds were resolved using a single column buffer system (2).

Periodate Oxidation of Cross-linking Compounds-The peptide sample (70 nmol) was dissolved in 0.5 ml of 0.2 M NaHCO$, pH 8.5, in a glass screw-cap vial. Periodic acid (Baker) was added to a concen- tration of 0.03 M. The reaction mixture was incubated for 24 h in the dark at 22°C. The entire solution was then adjusted to pH 5 and applied to a column (1 x 130 cm) of Bio-Gel P-10 equilibrated in 30% acetic acid to resolve the reaction mixture. Control studies in which the reduced intermolecular cross-link, hydroxylysinohydroxynorleu- tine, was incubated with periodic acid in several different buffers for varying lengths of time established the conditions used for treating the peptide. Maximal cleavage of the cross-link standard by this procedure was 75%.

Sequence Analysis-Manual sequence analysis was performed us- ing the manual dansyl’-Edman technique (18). Dansyl amino acids were identified by two-dimensional thin layer chromatography on polyamide thin layer sheets (Schleicher and Schuell) (19).

Automated sequence analysis was performed as previously reported (20) using a Beckman model 89OC Sequencer. PTH-amino acids were identified by two-dimensional thin layer chromatography on polyam- ide sheets using the solvent system described by Kulbe (21) and quantitated by amino acid analysis following back conversion of the derivatives to the corresponding amino acids by hydrolysis in hy- driodic acid vapor (54.8%, containing 0.08% hypophosphorous acid as a preservative, Fisher Scientific Co.) (20).

RESULTS

Insoluble calf bone collagen purified according to the method described has an amino acid composition character- istic of type I collagen derived from other sources (17), indi- cating a homogeneous preparation. Following reduction and labeling of the cross-links with [3H]NaBH4, the collagen had a specific radioactivity of 18,700 cpm/mg of protein. The tritium label was distributed among the reducible intermolec- ular cross-links hydroxylysinohydroxynorleucine, hydroxy- lysinonorleucine, lysinonorleucine, and histidinohydroxymer-

’ The abbreviations used are: dansyl, 5-dimethylaminonapbtha- lene-l-sulfonyl; PTH, phenylthiohydantoin.

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A Cross-linked Peptide from Calf Bone Collagen

B4

I3 (u b

$2 0

2

I

C

FRACTION NUMBER

-KY C DE GHI J

50 100 150 200

FRACTION NUMBER

FIG. 3. Ion exchange chromatography of peptide material eluting from Sephadex G-25 in Fraction A. A, Aminex A-5 (2.5 x 60 cm) in 0.2 M pyridine acetate, pH 3.5, at 50°C. Peptides were eluted with a linear gradient of 1600 ml from 0.2 M pyridine acetate, pH 3.5, to 0.8 M pyridine acetate, pH 5.2, at a flow rate of 100 ml/h. Fractions of 10 ml were collected and pooled as indicated by brachets. B, DEAE- Sephadex A-25 (1 X 23 cm) in 0.1 M Tris, pH 8.3. Peptides eluting in Fraction A (above) were eluted with a linear gradient of 500 ml from 0 to 0.4 M NaCl in 0.1 M Tris, pH 8.3. Fractions of 2.5 ml were collected at a flow rate of 35 ml/h. Absorbance, 230 nm (- - -), “H, counts per min (-).

odesmosine in a ratio of 4:2:1:2. Some radioactivity was incor- porated into the cross-link precursors allysine and hydroxy- allysine as well (22), but under the labeling conditions used no radioactivity was found in any other amino acids (17). Cyanogen bromide digestion of the reduced insoluble collagen was judged complete by quantitative conversion of methionine to homoserine.

The result of hydroxyapatite chromatography of the CNBr peptide mixture is shown in Fig. 1. The material eluted in two major peaks and one minor peak which have been partially characterized (15). Fraction 2, which was enriched in high molecular weight cross-linked peptides, was subjected to lim- ited alkaline hydrolysis followed by fractionation on a Seph- adex G-25 column, as shown in Fig. 2. The higher molecular weight Fraction A, containing 38% of the radioactive material, was then separated by cation exchange chromatography on Aminex A-5 in pyridine acetate buffers as described under “Experimental Procedures.” The resulting fractionation of tritium-labeled material is illustrated in Fig. 3A. As indicated, much of the radioactive material eluted from the column unretarded. The remaining material eluted in a large number of incompletely resolved peaks which, on subsequent analysis,

appeared to be free cross-links and peptides of 2 to 5 residues. The cross-link-containing peptide material which was not adsorbed by Aminex A-5 was fractionated using a DEAE- Sephadex column developed with a linear gradient in sodium chloride as indicated in Fig. 3B. Fractions were pooled as indicated, and Fraction G was re-chromatographed on a col- umn of Bio-Gel P-10 (Fig. 4A), where the cross-link-containing material was well separated from smaller nonradioactive pep- tides. The material in Fraction A was pooled and subsequently re-chromatographed on a Bio-Gel P-30 column (Fig. 4B) where it eluted in the fractionating range as a symmetrical peak. Fractions were pooled as indicated, and a portion of the material (3.5 nmol) was acid hydrolyzed and its amino acid and cross-link compositions were determined. These data are presented in Table I. By comparison with unfractionated calf bone collagen (17), this material was significantly enriched in glutamate, proline, leucine, phenylalanine, histidine, and the reduced intermolecular cross-links hydroxylysinohydroxynor- leucine and hydroxylysinonorleucine. It contained no hydrox- yproline, threonine, or tyrosine and had relatively low levels of glycine, alanine, and serine. Calculation of the number of residues per peptide, based on the assumption that it con- tained 1 residue of aspartic acid, indicated that the peptide consisted of 19 amino acid residues and one intermolecular cross-link which was incomnletelv hvdroxvlated. A total of 140 nmol of the peptide wasbbtained: ”

’ E

)I

>.

I.

3. C

\

1 50 100 150 2 FRACTION NUMBER

10

FIG. 4. Gel filtration chromatography of DEAE-Sephadex peptide Fraction G (Fig. 3B). A, Bio-Gel P-10 (1 x 130 cm) column in 10% acetic acid. Fractions of 1 ml were collected at a flow rate of 6 ml/h. B, Bio-Gel P-30 (0.8 X 100 cm column) in 30% acetic acid. Fractions of 0.5 ml were collected at 2 ml/h. C, rechromatography on Bio-Gel P-10 as in A following periodic acid cleavage of the cross-link. Brack- ets indicate fractions pooled. 3H counts per min, -; absorbance at 570 nm, ---.

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A Cross-linked Peptide from Calf Bone Collagen 669

TABLE I Amino acid composition of cross-linked peptide isolated following

Bio-Gel P-30 chromatography

Amino acids IlnlOl” Residues per thousand

Residues ter peptide

Hydroxyproline’ Aspartate 2.86 49.5 1.07 (1) Threonine 0.10 1.7 0.03 Serine 0.52 9.0 0.19 Glutamate 16.06 277.8 6.00 (6) Proline 13.69 236.8 5.11 (5) Glycine 5.42 93.8 2.02 (2) Alanine 3.04 52.6 1.13 (1) Valine 1.10 19.0 0.41 Isoleucine 0.28 4.8 0.10 Leucine 3.72 64.4 1.39 (1) Tyrosine’ Phenylalanine 3.21 55.5 1.20 (1) Hydroxylysine 0.94 16.3 0.35 Histidine 1.72 29.8 0.64 (1) Lysine 0.78 13.5 0.29 diOHLNL” 2.73 47.2 1.02 (1) OHLNL’ 0.88 29.8 0.33 (1)

57.05 (19)

U Peptide material was hydrolyzed in 6 N HCl for 24 h at 110°C. Average nanomoles derived from duplicate analyses. No corrections made for hydrolytic losses.

’ Based on 6 residues of glutamate per peptide. ’ Not detected. ” diOHLNL, hydroxylysinohydroxynorleucine; OHLNL, hydroxy-

lysinonorleucine.

In order to confirm the homogeneity of the preparation, a small fraction was analyzed by the manual dansyl-Edman technique. These results are presented in Table II. Two amino acids were apparent in each of the first two steps, indicating the presence of a double chain peptide.

In order to obtain a more quantitative sequence analysis, 40 nmol of the peptide material was subjected to five cycles of Edman degradation using the automated Sequencer. These results are also shown in Table II.

In order to determine the amino acid composition and sequences of the component polypeptide chains, the remaining material, approximately 70 nmol, was treated with periodic acid as described under “Experimental Procedures.” Periodic acid oxidation of hydroxylysinohydroxynorleucine proceeds according to the reaction scheme outlined in Fig. 5 and pri- marily results in selective cleavage of the cross-linked peptide at the cross-link residue.” The component chains were sepa- rated by chromatography on Bio-Gel P-10 as illustrated in Fig. 4C. Material from each of the fractions indicated was hydrolyzed, and the amino acid compositions were determined in duplicate (Table III). In addition, the elution positions of the tritium-containing components released upon acid hy- drolysis of both the intact peptide and the periodate cleavage products were determined by monitoring the effluent from the amino acid analyzer column. Fraction B (Fig. 4C) eluted from the P-10 column behind the residual uncleaved peptide (Fraction A) and showed decreased levels of aspartate, gluta- mate, proline, and glycine compared to Fraction A. The cross- link was no longer detectable as hydroxylysinohydroxynorleu- tine, and all of the radioactivity eluted at 72 min, in the position of serine. There was, however, no ninhydrin-positive peak eluting at this position. This is the known position of elution of a-aminoglutaric semialdehyde and its reduced de- rivative, hydroxynorvaline (7).

Fraction C contained an g-residue peptide whose composi- tion is shown in Table III. The value for proline content is

,’ Oxidation of other amino acid residues has been reported previ- ously (23) and was observed here as well.

TABLE II

Partial amino acid sequence of the intermolecular cross-linked peptide

TLC dansyl- Amino acids Residue amino acids identified after Yields TLC PTH-

HI hydrolysis” amino acids

1 Phe + Pro 2 Leu + Glu 3 Pro 4 Glu 5 -0

Phe + Pro Leu + Glu

Pro Glu Pro

nmol 35.9 + 10.6 31.1 + 18.4

28.9 20.4 13.5

Phe + Pro Leu + Glu

Pro Glu Pro

” Hydrolysis of the PTH-derivatives was performed as described under “Experimental Procedures.”

’ Not determined.

much lower than expected, probably because of destruction of NHi-terminal proline by periodate (24). The alanine content was higher than expected in both Fractions B and C, possibly due to partial conversion of several amino acids to compounds eluting at that position. The oxidized cross-link eluted at the position of serine as expected from a previous report (7). The sum of the compositions of the two peptides (Table III) is in agreement with that of the intact cross-link-containing pep- tide (Table I).

In order to obtain the maximum amount of sequence data from the larger peptide, all of the material which eluted in Fractions 104 to 130 (approximately 40 nmol), containing a mixture of the uncleaved starting material and the 12-residue peptide, was pooled and applied to the sequenator. It was anticipated that the contribution from the smaller peptide remaining attached to the uncleaved peptide in Fraction A would be decreased to a level which would not interfere with interpretations of the data while at the same time enriching the concentration of the peptide in Fraction B. The material was subjected to 10 cycles of Edman degradation and the PTH-amino acids at each step were identified by polyamide thin layer chromatography and quantitated by HI hydrolysis to the free amino acids followed by amino acid analysis (Table IV). The sequence beginning Pro-Glu observed with the intact peptide (Table II) was no longer observed. At residue 9 the yield dropped considerably and only 2.2 nmol of glutamate were recovered following HI hydrolysis. The PTH-derivative of residue 10 appeared as a very faint spot on the polyamide thin layer chromatogram, migrating to the position of PTH- lysine. An assay of the relative amount of tritum label ex- tracted with each sequential amino acid indicated that most of the peptide had been extracted from the Sequencer cup by residue 9.

The recovery of the smaller peptide following periodate cleavage was so low (less than 10 nmol) that additional se- quence data could not be obtained.

DISCUSSION

Limited alkaline hydrolysis of partially purified cyanogen bromide peptides obtained from [“H]NaBH4-reduced insolu- ble calf bone collagen yielded a homogeneous, double chain cross-linked peptide of 19 residues. The individual peptides contained 8 and 12 amino acids, respectively, and were cova- lently linked by hydroxylysinohydroxynorleucine, the pre- dominant reducible intermolecular cross-link of bone collagen. The amino acid sequence of the first 10 residues of the 12- residue peptide (Fig. 6) was identical with the published sequence of the homologous al chain of skin collagen encom- passing residues 8” to 17” with the exception that residues ll”, 14”, and 15” were originally identified as glutamine rather than glutamate as reported here. It should be noted, however, that the initial alkaline hydrolysis used in this procedure was

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670 A Cross-linked Peptide from Calf Bone Collagen

\/ (tH2) 2 CH-OH

CH 4 - ;H - I 2 CH-OH I

(CH2j2

A

Y (?2)2

CEO I

73 NH dH I 2

CH-OH I

(CH2) 2

A

Nd3H,

(tH2) 2 CH-OH

3HbH

NH I

73 CH-OH

(bH2) A

NH

CH I 2

H5106

H5106

Y (YH2) 2 HC=O + 3HCH0 +

NH3

Y (cH2)2

3H;=0 + H2C=0 + NH~

CH-OH I

(cH2)2

A

FIG. 5. Reaction products of hydroxylysinohydroxynorleucine following treatment with periodic acid.

TABLE III TABLE IV Amino acid composition of the peptide components obtained

followingperiodic acid cleavage of the intermolecular cross-link

Values were derived from duplicate analyses following hydrolysis in 6 N HCl for 24 h at 110°C.

Amino acid sequence of the first IO residues of the longer chain of the cross-linked peptide isolated following periodate cleavage of

the cross-link -Phe-Leu-Pro-Glu-Pro-Pro-Glu-Glu-Glu-Lys

Amino acid P-IO(R) RPP”

P~~$$’ P-10(R) + P-10(C)

Aspartate Serine (hydroxynorvaline) Glutamate Proline Glycine Alanine Leucine Phenylalanine Histidine diOHLNL OHLNL

0.04 (0) 0.07 (1) 4.00 (4) 3.47 (3) 0.69 (0) 2.27 (1) 0.87 (1) 0.65 (I) 0.14 (1) 0.09 0.02

(12)

0.72 (1) 1 0.76 (1) 2 2.00 (2) 6 0.44 (2) 5 2.02 (2) 2 0.90 (0) 1 0.26 (0) 1 0.24 (0) 1 0.17 (0) 1 -,’

, I

(8) 20

rr Residues per peptide based on 4 residues of glutamate per pep- tide.

’ Residues per peptide based on 2 residues of glutamate per peptide. Values in parentheses are the nearest integral values and include corrections for destruction due to periodic acid (23).

’ diOHLNL, hydroxylysinohydroxynorleucine; OHLNL, hydroxy- lysfyonorleucine.

-, not detected.

performed under conditions which resulted in deamidation. This peptide was therefore assigned to the COOH-terminal region, residues 8” to 19”. The occurrence of glycine in every third position throughout the helical region of type I collagen and the absence of a peptide with a similar composition in the NHz-terminal nonhelical extensions (4) rule out the possibility of assigning this peptide to any other sequence in the molecule. Because the peptide composition contained no aspartate after periodate cleavage and purification (Table III), it was con- cluded that the COOH-terminal residue was the histidine at position 19”, yielding the 12-residue peptide shown. The COOH-terminal histidine was destroyed by periodic acid oxi- dation as has previously been observed (24).

The sequence of the smaller, S-residue peptide was not determined beyond the first 2 residues because of poor recov- ery following periodate cleavage. In the intact peptide, this

Cycle Amino acids iden- tified after HI hy-

drolvsis” Yield TLC

nmol

1 Phe 12.3 PTH-Phe 2 Leu 14.0 PTH-Leu 3 Pro 14.0 PTH-Pro 4 Glu 11.5 PTH-Glu 5 Pro 9.2 PTH-Pro 6 Pro 8.2 PTH-Pro 7 Glu 7.2 PTH-Glu 8 Glu 5.6 PTH-Glu 9 -* PTH-Glu

10 -b PTH-Lys

D Hydrolysis of the PTH-derivatives was performed as described under “Experimental Procedures.”

b -. not detected.

al- lo= 15= ~phe-Leu-Pro-Glu-Pro-Pro-Glu-Glu-Glu-Hyl-Ala-His-

I -Pro-Clu, Gly, Hyl, Asp, Gly, Pro, Glu

FIG. 6. Proposed structure of the intermolecular cross-linked pep- tide.

sequence could not be detected after the 2nd residue. The reason for this is not known but may result from close prox- imity of the cross-link, which is not expected to yield a free PTH-derivative at this point. The behavior in the automated Sequencer of the remaining peptide residues beyond the cross- link is not known for collagen peptides and may be blocked because of steric restrictions imposed by the cross-link.

The glycine content of this peptide, however, strongly sug- gests that it arises from a helical region of the molecule. Furthermore, the dipeptide Pro-Glu occurs only a few times throughout the published sequences of type I collagen and published (Y chain compositions rule out all possible assign- ments except the one beginning at residue 662 in an a2 chain.

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A Cross-linked Peptide from Calf Bone Collagen

The sequence beyond residue 663, however, has not been Acknowledgments-We wish to thank Dr. Juris 0~01s and Craig

determined. Gerard for providing the automated sequence analyses.

The overlap distance between adjacent collagen molecules required by a cross-link between hydroxylysine residues at ~yl 17” and (~2 665 is not consistent with the overlaps predicted from the “quarter stagger” model for the collagen fibril (23), although there is no direct evidence indicating that this is a correct polymer model for calf bone collagen. Using the pre- viously reported cross-link between hydroxylysine at al 17“ and lysine at al 87 as the origin (7, lo), the quarter stagger hypothesis predicts that the smaller peptide must derive from the vicinity of residue 87, 327, or 564. Again, comparison by homology rules out every possible assignment but the area near residue 564 in the a2 chain for which sequence data are not available. It is also possible that the sequence of calf bone collagen is not homologous to that of calf skin. Thus, a definite assignment for the sequence of the 8-residue peptide cannot be made until the (Y chain sequence is reported in its entirety.

Following periodate cleavage of hydroxylysinohydroxynor- leucine and amino acid analysis of the component peptides, the radioactivity was partially retained by the larger peptide and was converted to a compound eluting in the position of [,‘H]hydroxynorvaline, which did not react with ninhydrin. The ninhydrin value of the oxidized cross-link associated with the smaller peptide was also lower than expected (40% of the expected value). In addition, some other amino acids, partic- ularly histidine and proline, were recovered in lower yields than expected, suggesting that periodate oxidation, under the conditions employed, alters several other amino acids in ad- dition to the cross-link. Amino acid oxidation by periodate has been reported previously (24). The fact that most of the “H label remained with the larger peptide indicates that the cross-link precursor, hydroxyallysine, derived from enzymatic conversion of the COOH-terminal hydroxylysine at position 17”, and condensed with the hydroxylysine residue on the other peptide. Since residue 17” has been found cross-linked to another residue at position 87 (7, 8) in addition to the one described here, it can evidently undergo cross-link formation with lysine or hydroxylysine residues in adjacent collagen molecules which are close enough to react.

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N P Stimler and M L Tanzerpeptide from insoluble calf bone collagen.

Isolation and characterization of a double chain intermolecular cross-linked

1979, 254:666-671.J. Biol. Chem. 

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