Identification of a Novel Splice Variant of Neural Cell Adhesion Molecule in Glioblastoma Through Proteogenomics Analysis

Abstract

Splice variants are known to be important in the pathophysiology of tumors, including the brain cancers. We applied a proteogenomics pipeline to identify splice variants in glioblastoma (GBM, grade IV glioma), a highly malignant brain tumor, using in-house generated mass spectrometric proteomic data and public domain RNASeq dataset. Our analysis led to the identification of a novel exon that maps to the long isoform of Neural cell adhesion molecule 1 (NCAM1), expressed on the surface of glial cells and neurons, important for cell adhesion and cell signaling. The presence of the novel exon is supported with the identification of five peptides spanning it. Additional peptides were also detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel separated proteins from GBM patient tissue, underscoring the presence of the novel peptides in the intact brain protein. The novel exon was detected in the RNASeq dataset in 18 of 25 GBM samples and separately validated in additional 10 GBM tumor tissues using quantitative real-time-polymerase chain reaction (qRT-PCR). Both transcriptomic and proteomic data indicate downregulation of NCAM1, including the novel variant, in GBM. Domain analysis of the novel NCAM1 sequence indicates that the insertion of the novel exon contributes extra low-complexity region in the protein that may be important for protein-protein interactions and hence for cell signaling associated with tumor development. Taken together, the novel NCAM1 variant reported in this study exemplifies the importance of future multiomics research and systems biology applications in GBM.

Molecular and Clinical Insights into the Invasive Capacity of Glioblastoma Cells

“…Furthermore, the expression of NCAMs is inversely related to glioma grade, which is in agreement with data showing that loss of this molecule enhances tumor cell migration [5]. Recent transcriptomic and proteomic analyses have reproduced these findings and have identified a new splice variant of NCAM1 with potential implications in cell signaling [6].…”

Proteogenomics Integrating Novel Junction Peptide Identification Strategy Discovers Three Novel Protein Isoforms of Human NHSL1 and EEF1B2

“…With the development of the Human Proteome Project, − massive high-quality MS data have accumulated, although not all have been fully exploited. Mining novel protein isoforms from MS data is currently done primarily by searching a three-frame translation of transcriptome data; , however, unannotated protein isoforms often lack known transcripts and homologous proteins. In such cases, traditional proteogenomics based on whole-genome six-frame translation cannot capture novel alternative splice sites or determine the complete sequence of novel protein isoforms. , …”