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Identification and Characterization of Two Distinct -(1–3)-L-Fucosyltransferase Activities in Human Colon Carcinoma

Departments of Cell Sciences [G. B. S., T. F. K., M. M. C.] and Analytical Chemistry [K. R. A.], SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406

Two distinct -(1–3)-fucosyltransferase activities have been identified in the colon carcinoma cell lines HT-29 and COLO-205. While both enzymatic activities exhibit similar affinities for a synthetic -(1–3) acceptor and GDP-fucose, they differ with respect to divalent cation requirements, N-ethylmaleimide inhibition, and glycoprotein substrate specificity. The COLO-205 -(1–3) activity exhibits maximal enzymatic activity in the presence of 20 mM Mn²⁺ but retains less than 10% activity in the absence of divalent cations. In contrast, the optimal Mn²⁺ concentration for the HT-29 enzyme is 1 mM, although this activity is relatively insensitive to divalent cation stimulation. In addition, the HT-29 -(1–3)-fucosyltransferase activity is resistant to inhibition by 30 mM N-ethylmaleimide and relatively inactive toward the glycoprotein substrate fetuin as compared to its desialylated derivative, asialofetuin. The COLO-205 activity is inhibited approximately 90% by N-ethylmaleimide and is equally active with either glycoprotein acceptor.

Although the -(1–3) specific activities are similar in both cell lines, N-ethylmaleimide-sensitive -(1–4) fucosyltransferase activity is 40-fold higher in COLO-205 as compared to HT-29, suggesting that the COLO-205 fucosyltransferase activity may be an -(1–3/4) enzyme, while the HT-29 activity appears to be an -(1–3) specific form.

Further examination of a panel of cell lines, tumor biopsies, and xenografts, based on the effect of metal ions and N-ethylmaleimide, indicated that both enzyme activities are similarly expressed in human colon carcinoma tissue.

¹ To whom requests for reprints should be addressed, at Department of Cell Sciences, L-109, SmithKline Beecham Pharmaceuticals, P.O. Box 1539, King of Prussia, PA 19406.

Received 6/ 5/90. Accepted 7/17/90.

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