Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Summary

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

Abstract

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.

Introduction

In the last two decades, several reports demonstrated the importance of post-translational modifications (PTM) in cellular development and several diseases like cancer, but recently protein lysine methylation has emerged as an another vital PTM. While initially histone lysine methylation was found to be an essential chromatin mark, later work also showed lysine methylation of several non-histone proteins ^1-4. The sequential transfer of methyl groups from S-adenosyl-L-methionine to the ε-amino group of lysine residues is catalyzed by a family of enzymes called Protein Lysine Methyltransferases (PKMTs) that contains over 60 proteins in the human genome. ....

Protocol

1. Preparation of Peptide Arrays

1. Programming of the Peptide Sequences
   1. Design peptide sequences for the synthesis using the MultiPep Spotter software. Enter the peptide sequences in single letter codes.
      Peptide 1: T A R K S T G G K A
   2. Generate alanine or arginine walk libraries with a specific command (.replace, A) to screen the important amino acids required for the recognition of a defined substrate. For instance in the following example the first line is the wild type sequence and from the second line each amino acid in a peptide is altered to alanine sequentially.
      replace, A
      T-A-R-K-S-T-G
      A

Results

Peptide arrays were successfully used to biochemically characterize the specificity of PKMTs, and several novel histone and non-histone substrates of PKMT were identified by this approach ^5,16-19. Defining the correct substrate spectrum of a PKMT (or any enzyme) is an essential step towards understanding of its molecular mechanism and cellular functions.

As an example of the application of the peptide SPOT array methylation method, we describe the results of the specificity analysis.......

Discussion

SPOT synthesis as described here is a powerful method to map protein-protein interaction sites and investigate the substrate recognition of peptide modifying enzymes. However, SPOT synthesis still has certain drawbacks, because although the peptides synthesized by the SPOT method are reported to have more than 90% purity ²¹ this is difficult to confirm in each instance. Therefore, results have to be reproduced by other methods for instance using protein domains or with purified peptides synthesized by standard.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ethanol abs Gradient Grade HPLC	Honeywell	10299901	Flammable
N,N-Dimethylformamide Peptide Synthesis	Biosolve	4193302	Flammable, Toxic
Piperidine ≥99% for Peptide Synthesis	Roth	A122.1	Flammable, Toxic, corrosive
Oxyma Pure (Ethyl (hydroxyimino)cyanoacetate )	Novabiochem	8510860100
N,N’-Diisopropylcarbodiimide purum ≥98% GC	Fluka	38370	Flammable, Toxic, corrosive
Acetic anhydride	Roth	CP28.1	Flammable, Toxic, corrosive
Triisopropylsilane 99%	Aldrich	233781	Flammable, Toxic
Dichlormethane ≥99.9%	Roth	P089.1	Carcinogenic
N-Methyl-2-Pyrrolidone	Roth	4306.2	Toxic
Derivatized cellulose Membrane	Intavis AG Köln	32.1
Trifluoroacetic acid	Roth	P088.2	Toxic, corrosive
Bromphenolblue	AppliChem	A3640.0010
Ammonium Hydrogen Carbonate	Roth	T871.2	Toxic
Sodium Dodecyl Sulfate Pellets	Roth	CN30.3	Toxic, Flammable
MultiPep Synthesizer	Intavis AG Köln	n.a.
HyperfilmTM high performance film	GE Healthcare	28906837
Phoretix software	TotalLab	n.a.