Dataset Information

Determination of carry-over contaminations in recombinant major peanut allergen Ara h 2 produced in Nicotiana benthamiana leaves and analysis of proline hydroxylation patterns

ABSTRACT: The major peanut allergen Ara h 2 undergoes site-specific hydroxylation at three proline residues within repetitive IgE-binding DPYSPOHS sequences. There has been no report of recombinant Ara h 2 production with hydroxyprolines until now. We produced recombinant Ara h 2 in N. benthamiana leaves to determine host-related contaminants and to analyze site-specific hydroxylation of proline residues.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Arachis Hypogaea Var. Vulgaris

SUBMITTER: Dorothea Anrather

LAB HEAD: Heimo Breiteneder

PROVIDER: PXD027015 | Pride | 2021-09-28

REPOSITORIES: pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	20200724_QExHFX1_RSLC1_Uezuelmez_Breiteneder_ext_Uni_MFPL_rArah2_50p.raw	Raw
	checksum.txt	Txt
	mqpar.xml	Xml
	target_rAra2.fasta	Fasta
	txt.zip	Other

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Publications

The Major Peanut Allergen Ara h 2 Produced in Nicotiana benthamiana Contains Hydroxyprolines and Is a Viable Alternative to the E. Coli Product in Allergy Diagnosis.

Üzülmez Öykü Ö Kalic Tanja T Mayr Vanessa V Lengger Nina N Tscheppe Angelika A Radauer Christian C Hafner Christine C Hemmer Wolfgang W Breiteneder Heimo H

Frontiers in plant science 20211004

Peanut allergy is a potentially life-threatening disease that is mediated by allergen-specific immunoglobulin E (IgE) antibodies. The major peanut allergen Ara h 2, a 2S albumin seed storage protein, is one of the most dangerous and potent plant allergens. Ara h 2 is posttranslationally modified to harbor four disulfide bridges and three hydroxyprolines. These hydroxyproline residues are required for optimal IgE-binding to the DPYSPOHS motifs representing an immunodominant IgE epitope ...[more]

PMID: 34671372

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Project description:The hydroxylation of proline residues to 4-trans-hydroxyproline (Hyp) is a common post-translational protein modification in plants, mediated by prolyl 4-hydroxylases (P4Hs). Hyps predominantly occur in a group of cell wall proteins, the Hydroxyproline-rich glycoproteins (HRGPs), where they are frequently O-glycosylated. While prolyl-hydroxylation and O-glycosylation are important, e.g. for cell wall stability, they are not desirable in plant-made pharmaceuticals. Sequence motifs recognized for prolyl-hydroxylation derived from vascular plants were proposed but did not include data from mosses, such as Physcomitrella. Here, a phylogenetic reconstruction of plant P4Hs identified six P4Hs in four subfamilies in mosses. We analysed the amino acid sequences and structural environments around Hyps in Physcomitrella utilizing 73 Hyp sites in 24 secretory proteins from multiple MS/MS datasets, and found that prolines in close proximity to other prolines, alanine, serine, threonine and valine were preferentially hydroxylated. About 95 % of the Hyp sites were predictable with a combination of previously defined motifs and methods. In our data, AOV (Ala-Hyp-Val) was the most frequent prolyl-hydroxylation pattern. Additionally, short arabinose chains were attached to Hyps in two cell-wall pectinesterases. A combination of 443 AlphaFold structure models and our MS data of peptides with nearly 3000 proline sites found Hyps predominantly on protein surfaces in disordered regions. Moss-produced human erythropoietin (EPO) exhibited plant-specific O-glycosylation with arabinose chains on two Hyps. This modification was significantly reduced in a P4H1 single knock-out (KO) Physcomitrella mutant. Quantitative proteomics after isotope labelling with different P4H-KO moss mutants revealed specific changes in the amount of proteins, including HRGPs, and a modified prolyl-hydroxylation pattern from the mutants, suggesting a differential function of the six Physcomitrella P4Hs. Quantitative RT-PCR proved a differential effect of single P4H KOs on the expression of the other five p4h genes, suggesting a partial compensation of the mutation. AlphaFold-Multimer models for Physcomitrella P4H1 and its target EPO peptide superposed with the crystal structure of Chlamydomonas P4H1 and a peptide substrate suggested significant amino acids in the active centre of the enzyme that form H-bonds with the peptide substrate, and revealed differences between P4H1 and the other five Physcomitrella P4Hs.

Dataset Information

Determination of carry-over contaminations in recombinant major peanut allergen Ara h 2 produced in Nicotiana benthamiana leaves and analysis of proline hydroxylation patterns

Dataset's files

Publications

The Major Peanut Allergen Ara h 2 Produced in <i>Nicotiana benthamiana</i> Contains Hydroxyprolines and Is a Viable Alternative to the <i>E. Coli</i> Product in Allergy Diagnosis.

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