7‐S Collagen: Characterization of an Unusual Basement Membrane Structure

Abstract

A new type of collageneous structure, tentatively named 7‐S collagen, was isolated from a mouse tumor basement membrane, mouse and human placenta, bovine lens capsule and human kidney. The protein was solubilized from the tissues by limited digestion with pepsin or trypsin and could easily be separated from other collageneous protein because of its resistence towards further degradation by bacterial collagenase at 20°C. 7‐S collagen showed an amino acid composition typical of basement membrane collagen and contained 22% carbohydrate mainly as glucosyl‐galactosyl bound to hydroxylysine but also some mannose and glucosamine. Ultracentrifugal analysis demonstrated that the proteins were homogeneous with a sedimentation coefficient of about 7.2 S and with a molecular weight of about 360000 both in phosphate buffer pH 7 and 6 M guanidine. The peptide was triple helical as shown by circular dichroism and exhibited a biphasic melting profile indicating two conformationally distinct domains with t_m= 48°C and 70°C. The more stable domain could be isolated as an homogeneous fragment (M_r= 225000) after a second digestion with collagenase at 37°C. This fragment contained all the disulfide bonds (42 Cys/1000 residues) of the original molecule. Electron microscopy showed a rod‐like structure in agreement with the hydrodynamic properties of 7‐S collagen. The dimensions of these peptides were 3 × 95 nm (long form) and 3.4 × 40–50 nm (short form). Complete reduction of 7‐S collagen under denaturing conditions produced several polypeptide chains in the molecular weight range of 27000–153000 which differ from each other by M_r increments 25000–27000. Separation of the chains on agarose did not reveal any simple stoichiometric relationship indicating that some chains are either cross‐linked or represent fragments produced during proteolytic treatments. Complete reduction of 7‐S collagen under non‐denaturing conditions lowered the thermal transition of the triple helix to 48°C but did not change its molecular weight except when exposed to dissociating solvents. 7‐S collagens were potent immunogens and could be characterized by radioimmunoassays. Antigenicity was slightly reduced by reduction and denaturation while collagenase at 37°C produced a larger decrease. Proteins obtained from various sources showed distinct immunological relationships although interspecies differences in affinity exist. No or only little cross‐reaction was observed with type IV and V collagens and some further fragments of basement membrane collagen. The data indicate that 7‐S collagen is a unique component of basement membranes which shows a more compact and stable structure than other collageneous proteins.