Characterization of Solanum tuberosum multicystatin and its structural comparison with other cystatins.

Mark S Nissen,W Jordan Ballinger,Ka Sum Lam,N Richard Knowles,Buhyun Youn,D Benjamin Knowles,Chulhee Kang,G.N Mohan Kumar

doi:10.1105/tpc.108.064717

Mark S Nissen, W Jordan Ballinger + Show 6 more

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https://doi.org/10.1105/tpc.108.064717

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Abstract

Potato (Solanum tuberosum) multicystatin (PMC) is a crystalline Cys protease inhibitor present in the subphellogen layer of potato tubers. It consists of eight tandem domains of similar size and sequence. Our in vitro results showed that the pH/PO(4)(-)-dependent oligomeric behavior of PMC was due to its multidomain nature and was not a characteristic of the individual domains. Using a single domain of PMC, which still maintains inhibitor activity, we identified a target protein of PMC, a putative Cys protease. In addition, our crystal structure of a representative repeating unit of PMC, PMC-2, showed structural similarity to both type I and type II cystatins. The N-terminal trunk, alpha-helix, and L2 region of PMC-2 were most similar to those of type I cystatins, while the conformation of L1 more closely resembled that of type II cystatins. The structure of PMC-2 was most similar to the intensely sweet protein monellin from Dioscorephyllum cumminisii (serendipity berry), despite a low level of sequence similarity. We present a model for the possible molecular organization of the eight inhibitory domains in crystalline PMC. The unique molecular properties of the oligomeric PMC crystal are discussed in relation to its potential function in regulating the activity of proteases in potato tubers.

Highlights

The soluble proteins of potato (Solanum tuberosum) are comprised primarily of protease inhibitor proteins (;50%) and patatin (;40%) (Pouvreau et al, 2001)
After 20 h of digestion, the 85-kD Potato (Solanum tuberosum) multicystatin (PMC) was reduced to three species of distinct molecular mass, ;35, ;15, and ;10 kD, which was maintained through 40 h of incubation
It has been reported that proteolytic fragmentation of PMC does not destroy its ability to inhibit papain, but the fragments no longer crystallize at basic pH and in the presence of phosphate (Walsh and Strickland, 1993)

Summary

Introduction

The soluble proteins of potato (Solanum tuberosum) are comprised primarily of protease inhibitor proteins (;50%) and patatin (;40%) (Pouvreau et al, 2001). The inhibitors include protease inhibitors I and II (and other Ser protease inhibitors), potato aspartate protease inhibitor, Kunitz-type protease inhibitor, potato carboxy-peptidase inhibitor, potato Cys protease inhibitor, and potato multicystatin (PMC) (Rodis and Hoff, 1984; Pouvreau et al, 2001). PMC is a Cys protease inhibitor (cystatin) that occurs in high concentrations as discrete crystals in the cortical parenchyma cells directly underneath the periderm of tubers (Hoff et al, 1972). In contrast with cystatins from other plant and animal sources, PMC has a high molecular mass (;85 kD/monomer); most plant protease inhibitors vary between 8 and 25 kD (Garcia-Olmedo et al, 1987). PMC can bind and inhibit several Cys proteases (e.g., papain) simultaneously, and the term multicystatin

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