Proteomic identification of arginine-methylated proteins in colon cancer cells and comparison of messenger RNA expression between colorectal cancer and adjacent normal tissues

Identification of type I protein arginine methyltransferase (PRMT) substrates and their functional significance during tumorigenesis is becoming more important. The present study aimed to identify target substrates for type I PRMT using 2-dimensional (2D) gel electrophoresis (GE) and 2D Western blot...

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Bibliographic Details
Published in:	Annals of coloproctology Vol. 38; no. 1; pp. 60 - 68
Main Authors:	Lim, Yongchul, Gang, Da Young, Lee, Woo Yong, Yun, Seong Hyeon, Cho, Yong Beom, Huh, Jung Wook, Park, Yoon Ah, Kim, Hee Cheol
Format:	Journal Article
Language:	English
Published:	Korea (South) Korean Society of Coloproctology 01-02-2022 대한대장항문학회
Subjects:	colorectal neoplasms mass spectrometry n,n-dimethylarginine Original protein-arginine n-methyltransferases two-dimensional gel electrophoresis 일반외과학 N,N-dimethylarginine Protein-arginine N-methyltransferases Colorectal neoplasms Mass spectrometry Two-dimensional gel electrophoresis
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Summary:	Identification of type I protein arginine methyltransferase (PRMT) substrates and their functional significance during tumorigenesis is becoming more important. The present study aimed to identify target substrates for type I PRMT using 2-dimensional (2D) gel electrophoresis (GE) and 2D Western blotting (WB). Using immunoblot analysis, we compared the expression of type I PRMTs and endogenous levels of arginine methylation between the primary colorectal cancer (CRC) and adjacent noncancerous tissues paired from the same patient. To identify arginine-methylated proteins in HCT116 cells, we carried out 2D-GE and 2D-WB with a type I PRMT product-specific antibody (anti-dimethyl-arginine antibody, asymmetric [ASYM24]). Arginine-methylated protein spots were identified by mass spectrometry, and messenger RNA (mRNA) levels corresponding to the identified proteins were analyzed using National Center for Biotechnology Information (NCBI) microarray datasets between the primary CRC and noncancerous tissues. Type I PRMTs and methylarginine-containing proteins were highly maintained in CRC tissues compared to noncancerous tissues. We matched 142 spots using spot analysis software between a Coomassie blue (CBB)-stained 2D gel and 2D-WB, and we successfully identified 7 proteins that reacted with the ASYM24 antibody: CACYBP, GLOD4, MAPRE1, CCT7, TKT, CK8, and HSPA8. Among these proteins, the levels of 4 mRNAs including MAPRE1, CCT7, TKT, and HSPA8 in CRC tissues showed a statistically significant increase compared to noncancerous tissues from patients using the NCBI microarray datasets. Our results indicate that the method shown here is useful in identifying arginine-methylated proteins, and significance of arginine modification in the proteins identified here should be further identified during CRC development.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	2287-9714 2287-9722
DOI:	10.3393/ac.2020.00899.0128