There is increasing evidence that inflammation may affect glycosylation and sulfation of various glycoproteins. The present study reports the effect of tumor necrosis factor alpha (TNF-alpha), a proinflammatory cytokine, on the glycosyl- and sulfotransferases of the human bronchial mucosa responsible for the biosynthesis of Lewis x epitope and of its sialylated and/or sulfated derivatives, which are expressed in human bronchial mucins. Fragments of macroscopically normal human bronchial mucosa were exposed to TNF-alpha at a concentration of 20 ng/ml. TNF-alpha was shown to increase alpha1,3-fucosyltransferase activity as well as expression of the two alpha1,3-fucosyltransferase genes expressed in the human airway, FUT3 and FUT4. It had no influence on alpha1,2-fucosyltransferase activity or FUT2 expression. It also increased alpha2,3-sialyltransferase activity and the expression of ST3Gal-III and, more importantly, ST3Gal-IV and both N-acetylglucosamine 6-O-sulfotransferase and galactose 3-O-sulfotransferase. These results are consistent with the observation of oversialylation and increased expression sialyl-Lewis x epitopes on human airway mucins secreted by patients with severe lung infection such as those with cystic fibrosis, whose airways are colonized by Pseudomonas aeruginosa. However, other cytokines may also be involved in this process.

A;lthough glycosphingolipids are ubiquitously expressed and essential for multicellular organisms, surprisingly little is known about their intracellular functions. To explore the role of glycosphingolipids in membrane transport, we used the glycosphingolipid-deficient GM95 mouse melanoma cell line. We found that GM95 cells do not make melanin pigment because tyrosinase, the first and rate-limiting enzyme in melanin synthesis, was not targeted to melanosomes but accumulated in the Golgi complex. However, tyrosinase-related protein 1 still reached melanosomal structures via the plasma membrane instead of the direct pathway from the Golgi. Delivery of lysosomal enzymes from the Golgi complex to endosomes was normal, suggesting that this pathway is not affected by the absence of glycosphingolipids. Loss of pigmentation was due to tyrosinase mislocalization, since transfection of tyrosinase with an extended transmembrane domain, which bypassed the transport block, restored pigmentation. Transfection of ceramide glucosyltransferase or addition of glucosylsphingosine restored tyrosinase transport and pigmentation. We conclude that protein transport from Golgi to melanosomes via the direct pathway requires glycosphingolipids.

Bronchial mucins were purified from the sputum of 14 patients suffering from cystic fibrosis and 24 patients suffering from chronic bronchitis, using two CsBr density-gradient centrifugations. The presence of DNA in each secretion was used as an index to estimate the severity of infection and allowed to subdivide the mucins into four groups corresponding to infected or noninfected patients with cystic fibrosis, and to infected or noninfected patients with chronic bronchitis. All infected patients suffering from cystic fibrosis were colonized by Pseudomonas aeruginosa. As already observed, the mucins from the patients with cystic fibrosis had a higher sulfate content than the mucins from the patients with chronic bronchitis. However, there was a striking increase in the sialic acid content of the mucins secreted by severely infected patients as compared to noninfected patients. Thirty-six bronchial mucins out of 38 contained the sialyl-Lewis x epitope which was even expressed by subjects phenotyped as Lewis negative, indicating that at least one α1,3 fucosyltransferase different from the Lewis enzyme was involved in the biosynthesis of this epitope. Finally, the sialyl-Lewis x determinant was also overexpressed in the mucins from severely infected patients. Altogether these differences in the glycosylation process of mucins from infected and noninfected patients suggest that bacterial infection influences the expression of sialyltransferases and α1,3 fucosyltransferases in the human bronchial mucosa.

UDP-galactose reaches the Golgi lumen through the UDP-galactose transporter (UGT) and is used for the galactosylation of proteins and lipids. Ceramides and diglycerides are galactosylated within the endoplasmic reticulum by the UDP-galactose:ceramide galactosyltransferase. It is not known how UDP-galactose is transported from the cytosol into the endoplasmic reticulum. We transfected ceramide galactosyltransferase cDNA into CHOlec8 cells, which have a defective UGT and no endogenous ceramide galactosyltransferase. Cotransfection with the human UGT1 greatly stimulated synthesis of lactosylceramide in the Golgi and of galactosylceramide in the endoplasmic reticulum. UDP-galactose was directly imported into the endoplasmic reticulum because transfection with UGT significantly enhanced synthesis of galactosylceramide in endoplasmic reticulum membranes. Subcellular fractionation and double label immunofluorescence microscopy showed that a sizeable fraction of ectopically expressed UGT and ceramide galactosyltransferase resided in the endoplasmic reticulum of CHOlec8 cells. The same was observed when UGT was expressed in human intestinal cells that have an endogenous ceramide galactosyltransferase. In contrast, in CHOlec8 singly transfected with UGT 1, the transporter localized exclusively to the Golgi complex. UGT and ceramide galactosyltransferase were entirely detergent soluble and form a complex because they could be coimmunoprecipitated. We conclude that the ceramide galactosyltransferase ensures a supply of UDP-galactose in the endoplasmic reticulum lumen by retaining UGT in a molecular complex.