Barley Lipid Transfer Protein, LTP1, Contains a New Type of Lipid-like Post-translational Modification*

Flemming M Poulsen,Peter Roepstorff,Kresten Lindorff-Larsen,Jakob R Winther,Mathilde H Lerche

doi:10.1074/jbc.m104841200

Abstract

In plants a group of proteins termed nonspecific lipid transfer proteins are found. These proteins bind and catalyze transfer of lipids in vitro, but their in vivo function is unknown. They have been suggested to be involved in different aspects of plant physiology and cell biology, including the formation of cutin and involvement in stress and pathogen responses, but there is yet no direct demonstration of an in vivo function. We have found and characterized a novel post-translational modification of the barley nonspecific lipid transfer protein, LTP1. The protein-modification bond is of a new type in which an aspartic acid in LTP1 is bound to the modification through what most likely is an ester bond. The chemical structure of the modification has been characterized by means of two-dimensional homo- and heteronuclear nuclear magnetic resonance spectroscopy as well as mass spectrometry and is found to be lipid-like in nature. The modification does not resemble any standard lipid post-translational modification but is similar to a compound with known antimicrobial activity.

Highlights

A group of proteins termed nonspecific lipid transfer proteins1 is found in plants
The function of plant nonspecific lipid transfer proteins (ns-LTPs) has for a long time been puzzling
The original suggestion that these proteins would act as intracellular lipid transporters was inconsistent with the observation that ns-LTPs are extracellularly located secretory proteins

Summary

EXPERIMENTAL PROCEDURES

Materials—Barley grains (cv optic) were purchased from Dansk Landbrugs Grovvareselskab. The dialyzed solution was centrifuged, and the pH in the supernatant was adjusted to 6.5 This solution was loaded onto a large CM-52 carboxymethyl cellulose column (2.6-cm internal diameter, 30 cm long) equilibrated with 20 mM potassium phosphate buffer, pH 6.5 (Buffer A). Relevant fractions were identified by SDS-PAGE and pooled, the pH was adjusted to 6.0, and the sample was loaded onto a smaller CM-52 column (1.6-internal diameter, 20 cm long) equilibrated with Buffer A. LTP1-containing fractions were pooled and applied, 8 ml at a time, to an S200 Sephacryl gel-filtration column (2.6-cm internal diameter, 60 cm long) equilibrated with 0.2 M (NH4)HCO3 (Buffer D). NMR Spectroscopy—ptm-LTP1 was dissolved in 600 ␮l of 90/10% H2O/D2O to a concentration of 3 mM All spectra on this sample were recorded on a Bruker AMX600 spectrometer at 14.1 tesla with a 5-mm triple-resonance probe. Spectra in negative ion mode were obtained under the same conditions as those in positive mode by switching all voltage polarities

RESULTS

Modified yЉ ion

DISCUSSION