780 Cell Biology international Reports, Vol. 4, No. 8,. August 1980

CROSS-LINKING OF COLLAGEN IN BASEMENT MPIBRANES

J.G. Heathcote*, A.J, Bailey+ & M.E, Grant* Dept. of Medical Biochemistry, Universit A.R.C. Meat Research Institute, 9

of Manchester* & Langford , U.K.

Basement membranes (BM) are relatively stable components of the extracellular matrix and have important morphogenetic and physiological functions. In vitro -- they appear to be quite insoluble in simple solvents such as 0.3M-acetic acid and phosphate-buffered saline but they can be partially solubilised by treatment with 8M-urea and mercaptoethanol indicating the presence of hydrogen bonds and disulphide bonds within the BM matrix.3

link(s) involved rat lenses were incubated with [4,%3H]lysine in lysine-free Dulbecco's modification of Eagle's medium for 24h. The capsules were then isolated, reduced with KBH4, hydrolysed in acid and analysed by ion-exchange chromatography. Radioactivity was detected in lysine, hydroxylysine and also dihydroxylysino- norleucine indicating that the cross-link was formed by the interaction of a hydroxyallysine (I) residue with a neighbouring hydroxylysine (II) residue (Fig.). Since the polymeric aggregates Isolated rat lens capsules were

incubated with [3H]proline for 2h in vitrowere not disrupted bv O.wacetic acid it -- and a collagenous polypeptide was is likely that-the &o&link (III) occurs

predominantly in the stable hydroxylysino- 5-ketonorleucine (IV) configuration.

Biosynthetic studies with embryonic-chick lenses and isolated rat renal glomeruli indicate that cross-linked aggregates are also formed in the BM's of these systems although not so readily as in the rat, lens capsule.2 Differences in the nature and number of cross-links in BM's may influence some of the properties of the membranes.

synthesised-which had--au-apparent mol. wt. of 18OK (estimated by SDS/agarose gel filtration chromatography after reduction and alkylation)l. On poly- acrylamide gels this polypeptide migrated slightly faster than the 8 units of type I collagen (mol. wt, 196K) but more slowly than the pro-a: chains (mol. wt. 15OK). When the radioactively-labelled lens-capsule polypeptides were analysed without prior reduction on a 5$ poly- acrylamide gel cross-linked with diallyl- tartardiamide a significant proportion of the radioactivity was found to have an electrophoretic mobility compatible with its being in a trimeric procollagen molecule. This result suggests that disulphide bonds are involved in the stabilization of BM procollagen.

The initially synthesised collagenous polypeptide was rapidly assembled into polymeric aggregates which were held together by hydroxylysine-derived cross- links. The formation of these aggregates was inhibited by the lathyrogen, p-amino- propionitrile.i To identify the cross-

OH I

;cH-cH2-cE2-CH-CEO

(1) ?H

1. Heathcote, J.G., Sear, C.H.J. & Grant, M.E. (1978) Biochem. J. 176, 283-294.

2. Heathcote, J.G., Sear, C.H.J. & Grant, M.E. (1979) Front. Matrix Biol. 7, 37-48.

3. Hudson, B.G. & Spiro, R.G. (1972) J. Biol. Chem. 247, 4229-4238.

OH

H2N- CH2 - h- CH2- CH2 - I&

(11) ' OH I / )cH-cH2-cH2-&I- CH=N-CH2-CH-CH2-t-X2-CH

(III) 0 \

\1 OH )cH-cH2-cH2- 'd - aI2 - NH - cH2 - &I - cH2 - cH2 - 4

(m

Fig. Formation of hydroxglysino+-ketonorleucine

0309-l 651/80/080780-01/$02.00~0 01980 Academic Press Inc. (London) Ltd.