Activation of beta1,3-N-acetylglucosaminyltransferase-2 (beta3Gn-T2) by beta3Gn-T8. Possible involvement of beta3Gn-T8 in increasing poly-N-acetyllactosamine chains in differentiated HL-60 cells

Abstract

Enzymatic activities of some glycosyltransferases are markedly increased via complex formation with other transferases or cofactor proteins. We previously showed that beta1,3-N-acetylglucosaminyltransferase-2 (beta3Gn-T2) and beta3Gn-T8 can form a heterodimer in vitro and that the complex exhibits much higher enzymatic activity than either enzyme alone (Seko, A., and Yamashita, K. (2005) Glycobiology 15, 943-951). Here we examined this activation and the biological significance of complex formation in differentiated HL-60 cells. beta3Gn-T2 and -T8 were co-immunoprecipitated from the lysates of both-transfected COS-7 cells, indicating their association in vivo. We prepared inactive mutants of both enzymes by destroying the DXD motifs. The mixture of mutated beta3Gn-T2 and intact beta3Gn-T8 did not exhibit any activation, whereas the mixture of intact beta3Gn-T2 and mutated beta3Gn-T8 had increased activity, indicating the activation of beta3Gn-T2 via complex formation. Next, we compared expression levels of beta3Gn-T1-T8 in HL-60 cells and DMSO-treated differentiated HL-60 cells, which produce larger poly-N-acetyllactosamine chains. The expression level of beta3Gn-T8 in the differentiated cells was 2.6-fold higher than in the untreated cells. Overexpression of beta3Gn-T8, but not beta3Gn-T2, induced an increase in poly-N-acetyllactosamine chains in HL-60 cells. These results raise a possibility that up-regulation of beta3Gn-T8 in differentiated HL-60 cells increases poly-N-acetyllactosamine chains by activating intrinsic beta3Gn-T2.

FIGURE 1.

In vivo interaction of wild-type and DXD-mutated β3Gn-T2 and β3Gn-T8. Co-immunoprecipitation of β3Gn-T2 and β3Gn-T8 (A), β3Gn-T2 and T8-QA (B), and T2-DA and β3Gn-T8 (C). COS-7 cells were transfected with expression vectors for the FLAG- or myc-tagged enzymes. Cell lysates were immunoprecipitated (IP) with anti-myc antibody. Equal aliquots of the pellets were analyzed by Western blotting (WB) with the antibodies indicated on the left.

FIGURE 2.

SDS-PAGE of soluble forms of β3Gn-T2 (T2), β3Gn-T8 (T8), and the mutated proteins, T2-DA and T8-QA, produced by P. pastoris. Purified proteins were applied to the gel. After electrophoresis, the proteins were detected by Sypro Orange staining. The digests by peptide:N-glycosidase F (PNGase F) are also shown in the right four lanes.

FIGURE 3.

Specific activities of β3Gn-T2 (T2), β3Gn-T8 (T8), T2-DA, T8-QA, and their combinations. Each protein (7 ng) was assayed for enzymatic activity as described under “Experimental Procedures.” Tetra-antennary N-linked oligosaccharide was used as an acceptor substrate. The enzymatic activities are the means of five independent experiments. The minimal detectable amount of the activity was 0.005 pmol/min.

FIGURE 4.

Stability of the enzymatic activities of human β3Gn-T2 (closed circle) and the mixture of human β3Gn-T2 and β3Gn-T8 (open circle). Equal amounts of enzyme proteins were incubated in 10 mm HEPES-NaOH, 0.15 m NaCl (pH 7.2) at 37 °C, and at the indicated time, aliquots of the enzyme solution were assayed for the β3Gn-T activity. Four independent experiments were performed.

FIGURE 5.

Reverse transcription-PCR analysis of expression of eight β3Gn-Ts (T1–T8) in HL-60 cells (U), and DMSO-treated differentiated HL-60 cells (D). The amounts of cDNAs were normalized by the expression levels of β-actin. cDNAs were detected and quantified by ethidium-bromide staining.

FIGURE 6.

Tomato-lectin blotting analysis of crude extracts of HL-60 cells (Nul) and HL-60 cells transfected with plasmids carrying β3Gn-T2 (T2), T2-DA, β3Gn-T8 (T8), and T8-QA. Cell-extract proteins (3 μg) were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane. Three or more repeating units of LacNAc were recognized by biotin-conjugated tomato lectin. One representative blot of four depicted is shown.

FIGURE 7.

A hypothetical schematic model for the increase in polyLacNAc chains during differentiation of HL-60 cells. In HL-60 cells, β3Gn-T2 is more highly expressed than β3Gn-T8, and some β3Gn-T2 (circles) is free from the complex with β3Gn-T8. After DMSO treatment, β3Gn-T8 is up-regulated. Newly synthesized β3Gn-T8 forms a complex with free β3Gn-T2 and activates β3Gn-T2 (hexagons). The arrow sizes indicate the intensity of the relative enzymatic activities.