Homologous Recombination of a Mutant Transgene

In view of the difficulties in the interpretation of the results from introduction of the collagenase-resistance Mut IV transgene, in our second strategy we employed targeted mu-tagenesis to introduce the same mutation into the endogenous Col1a-1 gene.33 In contrast to the transgenic mice, mice generated by the gene-targeting technology that carried the same mutation in their endogenous Col1a-1 gene (the mutant allele[r] of the Col1a-1 gene was named Col1a1tm1 Jae) developed normally to young adulthood and only then displayed alterations compatible with impaired collagen turnover. Nevertheless, collagen extracted from the skin, tendon and bone was not cleaved in the helical domain by human MMP-1 (collagenase-1) or murine or human MMP-13 (collagenase-3) at what was previously considered to be the single site at which these enzymes cleave undenatured type I collagen. An example is shown in Figure 10.3. We had demonstrated earlier that MMP-8 (neutrophil collagenase or collagenase-2) also did not cleave this mutant collagen produced by fibro-blasts in cell culture.31 Our data on the effects of collagenases on cleavage of triple helical collagen that contained the targeted mutations in the a1(I) chains around the helical cleavage site indicated that all three chains in the type I heterotrimer must have the proper cleavable

Fig. 10.3. Time course of cleavage at the helical and aminotelopeptide sites in mouse type I collagen by purified rat interstitial collagenase and human fibroblast (MMP-1) collagenase. (A) SDS-PAGE (5% acrylamide) showing reaction products after incubation with +/+ (Lanes 1-5 and 11-15) or r/r collagens (Lanes 6-10 and 16-20) and sufficient purified human fibroblast (MMP-1) collagenase (Lanes 1-10) or rat (lanes 11-20) interstitial collagenase to digest ~50% of the pepsinized +/+ collagen after 24 hr at 20oC. The substrates were collagen extracted from skin of +/+ mice with pepsin and from skin of r/r (Col1a1tm1Jae) mice in 0.5M acetic acid without pepsin as described. 6 Parent solutions containing enzyme and substrate were prepared at 0oC and incubated at 20oC to start the reaction. Aliquots calculated to contain 25 |ig of collagen were withdrawn and the reaction stopped by the addition of 1/10 vol of 500mM EDTA at the indicated intervals. After the reactions were stopped by the addition of EDTA to final concentration of 50mM, the samples were stored at 4oC until analysis by SDS-PAGE. (B) Rates of cleavage of helical region ( conversion of pepsinized +/+ type I collagen to Aa1[I] components) and aminoterminal region ( conversion of p components in acid-extracted r/- type I collagen to a1[I] chains) analyzed by densitometry of samples shown in (A).

Fig. 10.3. Time course of cleavage at the helical and aminotelopeptide sites in mouse type I collagen by purified rat interstitial collagenase and human fibroblast (MMP-1) collagenase. (A) SDS-PAGE (5% acrylamide) showing reaction products after incubation with +/+ (Lanes 1-5 and 11-15) or r/r collagens (Lanes 6-10 and 16-20) and sufficient purified human fibroblast (MMP-1) collagenase (Lanes 1-10) or rat (lanes 11-20) interstitial collagenase to digest ~50% of the pepsinized +/+ collagen after 24 hr at 20oC. The substrates were collagen extracted from skin of +/+ mice with pepsin and from skin of r/r (Col1a1tm1Jae) mice in 0.5M acetic acid without pepsin as described. 6 Parent solutions containing enzyme and substrate were prepared at 0oC and incubated at 20oC to start the reaction. Aliquots calculated to contain 25 |ig of collagen were withdrawn and the reaction stopped by the addition of 1/10 vol of 500mM EDTA at the indicated intervals. After the reactions were stopped by the addition of EDTA to final concentration of 50mM, the samples were stored at 4oC until analysis by SDS-PAGE. (B) Rates of cleavage of helical region ( conversion of pepsinized +/+ type I collagen to Aa1[I] components) and aminoterminal region ( conversion of p components in acid-extracted r/- type I collagen to a1[I] chains) analyzed by densitometry of samples shown in (A).

sequences.30,33 Even a single mutated a1(I) chain prevented the cleavage of the other two +/ + chains (i.e., another a1(I) chain and an a2(I) chain). The KM for the triple helical type I collagen molecules had been previously shown to be several orders of magnitude lower than that for the corresponding gelatins.28 It had been also proposed that collagenases are able to attack triple helical collagen because the region around the cleavage site is relatively poor in Pro (hydroxyproline [Hyp]) in the (-Gly-X-Y-) triplet-Y-position as seen in Figure 10.1 and since Pro/Hyp stabilize the triple helix, the Pro-poor area is susceptible to unwinding 39. In this regard, in preliminary results using circular dichroism (in collaboration with Dr. H.P. Bachinger Shriners Hospital, Portland, OR), we found that substituting Pro in only 2/333 triplets would have such a profound effect on structure.

Was this article helpful?

0 0

Post a comment