Inactivating germ-line and somatic mutations in polypeptide N-acetylgalactosaminyltransferase 12 in human colon cancers

Abstract

Aberrant glycosylation is a pathological alteration that is widespread in colon cancer, and usually accompanies the onset and progression of the disease. To date, the molecular mechanisms underlying aberrant glycosylation remain largely unknown. In this study, we identify somatic and germ-line mutations in the gene encoding for polypeptide N-acetylgalactosaminyltransferase 12 (GALNT12) in individuals with colon cancer. Biochemical analyses demonstrate that each of the 8 GALNT12 mutations identified inactivates the normal function of the GALNT enzyme in initiating mucin type O-linked protein glycosylation. Two of these inactivating GALNT12 mutations were identified as acquired somatic mutations in a set of 30 microsatellite stable colon tumors. Relative to background gene mutation rates, finding these somatic GALNT12 mutations was statistically significant at P < 0.001. Six additional inactivating GALNT12 mutations were detected as germ-line changes carried by patients with colon cancer; however, no inactivating variants were detected among cancer-free controls (P = 0.005). Notably, in 3 of the 6 individuals harboring inactivating germ-line GALNT12 mutations, both a colon cancer and a second independent epithelial cancer had developed. These findings suggest that genetic defects in the O-glycosylation pathway in part underlie aberrant glycosylation in colon cancers, and they contribute to the development of a subset of these malignancies.

Fig. 1.

Somatic mutations inactivating GALNT12 in V400 and V866 colon cancers. (A) DNA sequencing chromatograms depict GALNT12 missense mutations detected in the V400 and V866 colon cancer cell lines and their matched primary colon tumors, but not in matched normal colon tissue from these same individuals. Arrows indicate positions of mutations, with “mut” designating mutant alleles. The mutations shown were confirmed by sequencing individual cloned GALNT12 PCR products amplified from the V400 and V866 primary colon cancers. (B) Quantitation of enzymatic activity of mutant GALNT12 proteins. (Lower) Expression in transfection assays of epitope-tagged WT and mutant GALNT12 proteins as determined by Western blot analysis. (Upper) Matching quantitation of the specific enzyme activities normalized to the activity of the WT protein. Each bar represents the average activity values from 3 independent biological replicates (see Materials and Methods). The error bars represent the SEM. Note the loss of functional activity of the 2 somatic GALNT12 mutants (<3%) compared with the WT protein.

Fig. 2.

Functional characterization of germ-line GALNT12 variants. Shown are the GALNT12 enzyme activities of variants identified exclusively in the germ lines of individuals with colon cancer (Cases), versus that of variants found among controls (Controls) (Table S1). (Lower) Expression in transfection assays of epitope-tagged GALNT12 proteins. In each experiment, the variant and WT GALNT12 proteins were assayed in parallel transfections. (Upper) Matching quantitation of the specific enzyme activities of GALNT12 variants normalized to the activity of the WT protein. Noted for each variant is the amino acid alteration. Note the marked loss of functional activity of the cancer specific variants compared with variants carried by controls (P = 0.001). The error bars represent the SEM. The nucleotide change for each variant and the protein domain location of these variants are listed in Table S1.

Fig. 3.

Structural mapping of inactivating GALNT12 mutations. Colon cancer associated mutations inactivating GALNT12 are depicted by homology mapping to the X-ray crystal structure of GALNT2 bound to an EA2 substrate peptide (EA2) and to UDP (15). The upper right domain is the lectin domain, whereas the lower left domain is the catalytic domain. Inactivating germ-line mutations are in orange, and somatic mutations in red. Residues in parenthesis designate the position of the homologous GALNT2 residues. The GALNT2 X-ray crystal structure does not include the first 75 residues of the N-terminal transmembrane and stem domains; thus, the label for the GALNT12 start codon mutation (M1I, ATG>ATA) is placed to the left of the crystal structure.