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Updated: May 1, 2026
Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics
Published on: October 13, 2020
Identification and characterization of a QM protein as a possible peptidoglycan recognition protein (PGRP) from the
Attasit Udompetcharaporn1, Kingkamon Junkunlo2, Saengchan Senapin3
1Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand; Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand.
Abstract:
In an attempt to identify a peptidoglycan recognition protein (PGRP) in Penaeus (Penaeus) monodon, in vitro pull-down binding assays were used between shrimp proteins and purified peptidoglycan (PG). By gel electrophoresis and mass spectrometry followed by Mascot program analysis, proteins from shrimp hemocyte peripheral membrane proteins showed significant homology to records for a QM protein, actin and prophenoloxidase 2 precursor (proPO2), while proteins from cell-free plasma showed significant homology to records for a vitellogenin, a fibrinogen related protein (FREP) and a C-type lectin. Due to time and resource limitations, specific binding to PG was examined only for recombinant PmQM protein and PmLec that were synthesized based on sequences reported in the Genbank database (accession numbers FJ766846 and DQ078266, respectively). An in vitro assay revealed that hemocytes would bind with and encapsulate agarose beads coated with recombinant PmQM (rPmQM) or rPmLec and that melanization followed 2h post-encapsulation. ELISA tests confirmed specific binding of rPmQM protein to PG. This is the first time that PmQM has been reported as a potential PGRP in shrimp or any other crustacean. The two other potential PGRP identified (FREP and the vitellin-like protein present in male P. monodon, unlike other vitellin subunits) should also be expressed heterologously and tested for their ability to activate shrimp hemocytes.
Insights
This study identifies a novel peptidoglycan recognition protein (PGRP) in shrimp, PmQM, which binds to peptidoglycan (PG). This discovery advances our understanding of crustacean immune responses and potential PGRPs.
Area of Science:
- Marine Biology
- Immunology
- Biochemistry
Background:
- Peptidoglycan recognition proteins (PGRPs) are crucial in innate immunity.
- Identifying PGRPs in crustaceans like Penaeus monodon is essential for understanding their immune defenses.
Purpose of the Study:
- To identify and characterize peptidoglycan recognition proteins (PGRPs) in the shrimp Penaeus monodon.
- To investigate the binding capabilities of identified shrimp proteins with peptidoglycan (PG).
Main Methods:
- In vitro pull-down binding assays using shrimp proteins and purified peptidoglycan (PG).
- Gel electrophoresis, mass spectrometry, and Mascot program analysis for protein identification.
- Synthesis of recombinant PmQM and PmLec proteins for specific binding assays (ELISA).
Main Results:
- Shrimp hemocyte proteins showed homology to QM protein, actin, and proPO2.
- Plasma proteins were homologous to vitellogenin, fibrinogen-related protein (FREP), and C-type lectin.
- Recombinant PmQM (rPmQM) and PmLec (rPmLec) demonstrated binding to PG, with rPmQM showing specific binding via ELISA.
- Hemocytes encapsulated agarose beads coated with rPmQM or rPmLec, followed by melanization.
Conclusions:
- PmQM is identified as a potential PGRP in shrimp, marking a significant discovery in crustacean immunology.
- FREP and a vitellin-like protein are also suggested as potential PGRPs requiring further investigation.
- This research provides foundational insights into the molecular mechanisms of shrimp innate immunity against bacterial pathogens.
