Purification of keratan sulfate proteoglycan from monkey cornea

An explant culture of 15 cynomolgus monkey corneas was incubated with [35S]sulfate and [2-3H]mannose as labeling precursors. A 4 M guanidine HCl extract of the corneal stromas was prepared and combined with a 4 M guanidine HCl extract of stromas from 300 unlabeled corneas. The keratan sulfate proteoglycans in the combined extracts were purified by a combination of DEAE-cellulose chromatography, chondroitinase ABC digestion to remove chondroitin-dermatan sulfate proteoglycans, and elution from immobilized concanavalin A. The purified keratan sulfate proteoglycan was digested with papain and the digest was eluted on DEAE-Sephacel. The unbound fraction contained 59% of the 3H activity and consisted of intact oligosaccharide-peptides. The bound fraction, consisting of keratan sulfate chains linked to peptides, eluted during a linear 0-0.75 M NaCl gradient as a peak centered at approximately 0.6 M NaCl and contained 41% of the 3H and all of the 35S activity in the original proteoglycan. The chains were digested with endo-beta-galactosidase, and the digest was eluted on DEAE-Sephacel with a linear 0-0.75 M NaCl gradient. Most of the sulfated digestion fragments from the chains eluted as several distinct peaks during the gradient. All the 3H activity eluted in the unbound volume along with a small proportion of 35S activity. This unbound fraction was eluted on Bio-Gel P-10 to give a 3H peak (Kav = 0.46) well resolved from the remaining 35S activity which eluted near the total volume. This 3H peak contained the oligosaccharide-peptides derived from the linkage region between the keratan sulfate chains and the core protein. Structural analyses of the linkage region oligosaccharides and the intact oligosaccharides (Nilsson, B., Nakazawa, K., Hassell, J.R., Newsome, D.A., and Hascall, V.C. (1983) J. Biol. Chem. 258, 6056-6063) in combination with the 3H-labeling data suggest that the intact keratan sulfate proteoglycans contain an average of about one intact oligosaccharide per keratan sulfate linkage site.