KS-chain length, lumican expression, and galactose sulfation patterns in WT, htz, and null tissues were investigated using extracted corneal protein for Western blot analyses. Three samples were prepared for each genotype: untreated, PNGase F digested, and endo-β-galactosidase digested. The untreated samples, prepared to determine intact KS-GAG chain length as a function of the rate at which lumican migrated through the electrophoretic gel, revealed that WT and htz produced similar high-to-low molecular weight smear patterns, ranging from ∼160 to 45 kDa (
Fig. 3, lanes 1 and 2, respectively). In contrast,
B3gnt7-null corneal extracts showed a more condensed band from ∼65 to 45 kDa (
Fig. 3, lane 3), suggesting that lumican from
B3gnt7-null extracts may be substituted with lower-molecular-weight (i.e., shorter) KS-GAGs compared with WT or htz samples. To confirm that this finding was a consequence of KS-GAG chain length, PNGase F was used to cleave KS side chains from their lumican core proteins. After PNGase F predigestion, all three samples showed identical narrow bands at ∼40 kDa (
Fig. 3, lanes 4–6), which corresponds to the known molecular weight of lumican, ∼36 to 40 kDa.
29 Endo-β-galactosidase (endo-β-gal-ase) treatment was used to probe for aberrant galactose sulfation of KS-GAGs, as its cleavage site is the β-1,4 glycosidic linkage between unsulfated galactose and GlcNAc (sulfated or unsulfated).
30 It should be noted that endo-β-gal-ase will cleave at each unsulfated galactose along KS-GAGs, leaving lumican core proteins bound to the KS linkage region and GAG stub proximal to the innermost unsulfated galactose. Therefore, because WT, htz, and null samples that were digested with this enzyme produced bands of comparable molecular weight and density at ∼47 kDa (
Fig. 3, lanes 7–9), it was inferred that the innermost unsulfated galactose was not atypically oversulfated in
B3gnt7-htz and -null mutants. Whether or not galactose residues in the non-reducing terminal region of KS are affected by the
B3gnt7 mutation remains unknown.