The rate of kallikrein cleavage of CHO cell–expressed Lewis HK light chain cultured in the absence and presence of tunicamycin. Purified CHO-expressed (His)6-tagged Lewis HK4-6 cultured in the absence (–) or presence (+)of tunicamycin were subjected to time-dependent cleavage by kallikrein. (A) Protein-stained gels indicate a more rapid disappearance of a 40-kDa band of the recombinant (His)6–HK4-6 in the absence of tunicamycin and a less rapid disappearance of a 37-kDa (His)6–HK4-6 band as it is cleaved by kallikrein. std indicates the position of the 39-kDa gel standard. (B) Quantitative densitometric analysis of the Lewis (His)6–HK4-6. The rate of cleavage as a function of time was analyzed. Cultured in the absence of tunicamycin (○), and in the presence of tunicamycin (•). Mean ± SEM, n = 3. The Lewis recombinant (His)6-tagged HK cleavage with the additional N-linked carbohydrate was significantly faster than that observed in its absence. Quantification of the data of 3 separate experiments of each recombinant (bottom) indicated significant slowing to a normal rate of cleavage (as observed for the Fischer and Buffalo rat products, Figure 7). This experiment indicates that the presence of the N-linked carbohydrate at amino acid 511 alters the susceptibility of the recombinant protein to kallikrein cleavage. It is not consistent with the hypothesis that the amino acid change alone or the missing O-linked carbohydrate in the light chain is the cause of the observed phenomena.
