Quantification of extracellular UDP-galactose

Abstract

The human P2Y(14) receptor is potently activated by UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP-GlcNAc), and UDP-glucuronic acid. Recently, cellular release of UDP-Glc and UDP-GlcNAc has been reported, but whether additional UDP-sugars are endogenous agonists for the P2Y(14) receptor remains poorly defined. In the present study, we describe an assay for the quantification of UDP-Gal with subnanomolar sensitivity. This assay is based on the enzymatic conversion of UDP-Gal to UDP, using 1-4-beta-galactosyltransferase. UDP is subsequently phosphorylated by nucleoside diphosphokinase in the presence of [gamma-(32)P]ATP and the formation of [gamma-(32)P]UTP is monitored by high-performance liquid chromatography. The overall conversion of UDP-Gal to [gamma-(32)P]UTP was linear between 0.5 and 30 nM UDP-Gal. Extracellular UDP-Gal was detected on resting cultures of various cell types, and increased release of UDP-Gal was observed in 1321N1 human astrocytoma cells stimulated with the protease-activated receptor agonist thrombin. The occurrence of regulated release of UDP-Gal suggests that, in addition to its role in glycosylation reactions, UDP-Gal is an important extracellular signaling molecule.

Figure 1. Enzymatic conversion of UDP-[³H]Gal to [³H]lactose

A, HPLC tracings illustrating the conversion of UDP-[³H]Gal to [³H]lactose ([³H]Lac) in the presence (bottom) or in the absence (top) of 4βGT (0.002 U/ml, 60 min, 30°C). B, incubations were for 60 min at 30°C in the presence of the indicated amount of 4βGT. C, the time-course of the reaction was examined using 0.002 U/ml 4βGT. D, the conversion of UDP-[³H]Gal to [³H]lactose was assessed in the absence (-) or presence (+) of 0.2 mg/ml α-lactalbumin. All reactions were performed in 100 μl DMEM-H containing 20 mM glucose, 100 nM (0.1 μCi) [³H]UDP-Gal, 5 mM MnCl₂, and (except as indicated in D) 0.2 mg/ml α-lactalbumin. Radioactive species were separated by HPLC. The data represent the mean ± SD from at least three independent experiments performed in duplicates.

Figure 2. 4βGT-catalized formation of lactose or N-acetyllactosamine

A, UDP-Gal but not UDP-Glc is a substrate of 4βGT. Incubations were as in Figure 1A, in the presence of the indicated concentration of UDP-[³H]Gal or UDP-[³H]Glc. B, effect of N-acetylglucosamine concentration on the conversion of 100 nM (0.1 μCi) UDP-[³H]Gal to [³H]N-acetyllactosamine. Radioactive species were separated by HPLC. The data represent the mean ± SD, n ≥ 3.

Figure 3. Assessing UDP-Gal via UDP phosphorylation

The conversion of [γ³²P]ATP to [γ³²P]UTP was assessed by HPLC, as detailed in Methods. A, UDP (10 nM) was incubated in the presence of 100 nM (0.2 μCi) [γ³²P]ATP and the indicated amount of NDPK. B, time course of the NDPK (0.1 U/ml)-catalyzed phosphorylation of 10 nM UDP in the presence of 100 nM (0.2 μCi) [γ³²P]ATP. C, the UDP calibration curve was performed in the presence of 0.1 U/ml NDPK and 100 nM (0.2 μCi) [γ³²P]ATP (5 min, 30°C). The reaction was lineal between 0–30 nM UDP (r² = 0.9996), displaying a slope a value of 0.93784 and an intercept b value of 0.07. D, UDP-Gal (at the indicated concentration) was incubated for 30 min in the absence or presence of 0.002 U/ml 4βGT. At the end of this period, NDPK and [γ³²P]ATP were added for 5 min, as detailed in C. The reaction was lineal between 0–30 nM UDP-Gal (a = 1.016, b = 0, and r² = 0.9998). The data represent the mean ± SD, n ≥ 4. All incubations were performed in 100 μL DMEM-H supplemented with MnCl₂ and α-lactalbumin, as indicated in Figure 1. Background radioactivity (i.e., observed in the absence of UDP-Gal and UDP) was subtracted from all data points to allow regression lines fitting to origin.

Figure 4. Enhanced UDP-Gal and UDP-Glc release from thrombin-stimulated 1321N1 human astrocytoma cells

A, cells were rinsed and pre-incubated in 300 μl DMEM-H for 1 h, and thrombin (20 nM) was subsequently added for 5 min. UDP-Gal and UDP-Glc present in the extracellular medium were quantified, as described in Methods. The results represent the main value (± SD) from two independent experiments performed in triplicate; (*) indicates significant differences relative to control (p < 0.01, t-test). B, the stability of extracellular UDP-Gal and UDP-Glc on 1321N1 cells was assessed in cultures spiked with 0.5 μCi of the indicated radiotracer. Samples were collected at the indicated times and the resulting [³H]species were analyzed by HPLC, as described in Methods (mean ± SD, n = 4).