Recombinant Expression And Functional Analysis Of Silkworm PGRP-S5

Recognition by Pattern recognition receptor（sPRRs）and Pathogen associated molecularpatterns（PAMPs） of invading microbes initiated innate immunity of invertebrate. In insectand other arthropod, Peptidoglycan recognition proteins （PGRPs） detect peptidoglycan （PG）of bacterial cell wall, leading to the activation of defense responses. Studies on functions ofPGRPs in the fruit fly （Drosophila melanogaster） is more, but it is relatively rare in thesilkworm （Bombyx mori）. There are12PGRPs belongs to PGRP family in silkworm, however,functions of most of these PGRPs are not clear. In this study, we cloned the PGRP-S5activitydomain and carried out studies on its functions in silkworm immune system.1. We found PGRP-S5transcription was up-regulated in the fat body and the midgut byPseudomonas aeruginosa or Staphylococcus aureus infection at different time point bysemi-quantitative RT-PCR, but the change in hemocytes was not significant. It implied thatPGRP-S5may be involved in the regulation of the innate immunity.2. Through bioinformatics analysis, the amino acid sequence of PGRPs fromHelicoverpa armigera、Samia cynthia ricini、Manduca sexta、Drosophila melanogaster、Galleria mellonella、Anopheles gambiae、Tribolium castaneum and T7lysozyme have highsimilarity with silkworm PGRP-S5and sequence alignment indicates that the five conservedresidues critical for zinc binding and PG interaction in Drosophila PGRP-LB are all present insilkworm PGRP-S5.3. We cloned the activity domain of silkworm PGRP-S5into pSMF expression vector,expressed the recombinant protein in E. coli. Sequence analysis showed that the length of thedomain’s cDNA is387bp and encodes135amino acids. The target protein PGRP-S5wasobtained by SUMO protease digestion from the vector protein, and both of the purificationwas used Ni-NTA column.4. Using recombinant PGRP-S5activity domain protein and purified peptidoglycans （PG）from different bacteria, we showed PGRP-S5activity domain protein has strong affinity toDAP-type PG from gram-positive bacteria, weak affinity or no binding to Lys-type PG fromgram-positive bacteria, and no binding to DAP-type PG from gram-negative bacteria. Nextwe used two kinds of bacteria for agglutination tests, and results revealed that PGRP-S5binds to PG from certain bacterial strains and induces bacteria agglutination.5. In the presence of Zn²⁺, PGRP-S5activity domain protein is an amidase, cleavagesDAP-type PG from B. subtilis and M. luteus, but it shows no amidase activity to PG from B.megaterium and E. coli. Protein of PGRP-S5activity domain strongly inhibites the growth ofboth gram-positive bacteria and gram-negative bacteria with presence of Zn²⁺.6. In addition, we manifested the specific recognition of PG by PGRP-S5activitydomain protein is involved in phenoloxidase activation pathway.In summary, our study revealed silkworm PGRP-S5activity domain protein is involvedin immunity and functions as a receptor for phenoloxidase and as an effecter to inhibitorbacterial growth.