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An extended AE-rich N-terminal trunk in secreted pineapple cystatin enhances inhibition of fruit bromelain and is posttranslationally removed during ripening.
Plant Physiol 2009; 151(2):515-27PP

Abstract

Phytocystatins are potent inhibitors of cysteine proteases and have been shown to participate in senescence, seed and organ biogenesis, and plant defense. However, phytocystatins are generally poor inhibitors of the cysteine protease, bromelain, of pineapple (Ananas comosus). Here, we demonstrated that pineapple cystatin, AcCYS1, inhibited (>95%) stem and fruit bromelain. AcCYS1 is a unique cystatin in that it contains an extended N-terminal trunk (NTT) of 63 residues rich in alanine and glutamate. A signal peptide preceding the NTT is processed in vitro by microsomal membranes giving rise to a 27-kD species. AcCYS1 mRNA was present in roots and leaves but was most abundant in fruit. Using immunofluorescence and immunoelectron microscopy with an AcCYS1-specific antiserum, AcCYS1 was found in the apoplasm. Immunoblot analysis identified a 27-kD protein in fruit, roots, and leaves and a 15-kD species in mature ripe fruit. Ripe fruit extracts proteolytically removed the NTT of 27-kD AcCYS1 in vitro to produce the 15-kD species. Mass spectrometry analysis was used to map the primary cleavage site immediately after a conserved critical glycine-94. The AE-rich NTT was required to inhibit fruit and stem bromelain (>95%), whereas its removal decreased inhibition to 20% (fruit) and 80% (stem) and increased the dissociation equilibrium constant by 1.8-fold as determined by surface plasmon resonance assays. We propose that proteolytic removal of the NTT results in the decrease of the inhibitory potency of AcCYS1 against fruit bromelain during fruit ripening to increase tissue proteolysis, softening, and degradation.

Authors+Show Affiliations

Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, Hawaii 96822, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

19648229

Citation

Neuteboom, Leon W., et al. "An Extended AE-rich N-terminal Trunk in Secreted Pineapple Cystatin Enhances Inhibition of Fruit Bromelain and Is Posttranslationally Removed During Ripening." Plant Physiology, vol. 151, no. 2, 2009, pp. 515-27.
Neuteboom LW, Matsumoto KO, Christopher DA. An extended AE-rich N-terminal trunk in secreted pineapple cystatin enhances inhibition of fruit bromelain and is posttranslationally removed during ripening. Plant Physiol. 2009;151(2):515-27.
Neuteboom, L. W., Matsumoto, K. O., & Christopher, D. A. (2009). An extended AE-rich N-terminal trunk in secreted pineapple cystatin enhances inhibition of fruit bromelain and is posttranslationally removed during ripening. Plant Physiology, 151(2), pp. 515-27. doi:10.1104/pp.109.142232.
Neuteboom LW, Matsumoto KO, Christopher DA. An Extended AE-rich N-terminal Trunk in Secreted Pineapple Cystatin Enhances Inhibition of Fruit Bromelain and Is Posttranslationally Removed During Ripening. Plant Physiol. 2009;151(2):515-27. PubMed PMID: 19648229.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - An extended AE-rich N-terminal trunk in secreted pineapple cystatin enhances inhibition of fruit bromelain and is posttranslationally removed during ripening. AU - Neuteboom,Leon W, AU - Matsumoto,Kristie O, AU - Christopher,David A, Y1 - 2009/07/31/ PY - 2009/8/4/entrez PY - 2009/8/4/pubmed PY - 2009/12/16/medline SP - 515 EP - 27 JF - Plant physiology JO - Plant Physiol. VL - 151 IS - 2 N2 - Phytocystatins are potent inhibitors of cysteine proteases and have been shown to participate in senescence, seed and organ biogenesis, and plant defense. However, phytocystatins are generally poor inhibitors of the cysteine protease, bromelain, of pineapple (Ananas comosus). Here, we demonstrated that pineapple cystatin, AcCYS1, inhibited (>95%) stem and fruit bromelain. AcCYS1 is a unique cystatin in that it contains an extended N-terminal trunk (NTT) of 63 residues rich in alanine and glutamate. A signal peptide preceding the NTT is processed in vitro by microsomal membranes giving rise to a 27-kD species. AcCYS1 mRNA was present in roots and leaves but was most abundant in fruit. Using immunofluorescence and immunoelectron microscopy with an AcCYS1-specific antiserum, AcCYS1 was found in the apoplasm. Immunoblot analysis identified a 27-kD protein in fruit, roots, and leaves and a 15-kD species in mature ripe fruit. Ripe fruit extracts proteolytically removed the NTT of 27-kD AcCYS1 in vitro to produce the 15-kD species. Mass spectrometry analysis was used to map the primary cleavage site immediately after a conserved critical glycine-94. The AE-rich NTT was required to inhibit fruit and stem bromelain (>95%), whereas its removal decreased inhibition to 20% (fruit) and 80% (stem) and increased the dissociation equilibrium constant by 1.8-fold as determined by surface plasmon resonance assays. We propose that proteolytic removal of the NTT results in the decrease of the inhibitory potency of AcCYS1 against fruit bromelain during fruit ripening to increase tissue proteolysis, softening, and degradation. SN - 0032-0889 UR - https://www.unboundmedicine.com/medline/citation/19648229/An_extended_AE_rich_N_terminal_trunk_in_secreted_pineapple_cystatin_enhances_inhibition_of_fruit_bromelain_and_is_posttranslationally_removed_during_ripening_ L2 - http://www.plantphysiol.org/cgi/pmidlookup?view=long&pmid=19648229 DB - PRIME DP - Unbound Medicine ER -