Switching Amino-terminal Cytoplasmic Domains of α(1,2)Fucosyltransferase and α(1,3)Galactosyltransferase Alters the Expression of H Substance and Galα(1,3)Gal

Abstract

When ␣(1,2)fucosyltransferase cDNA is expressed in cells that normally express large amounts of the terminal carbohydrate Gal␣(1,3)Gal, and therefore the ␣(1,3)galactosyltransferase (GT), the Gal␣(1,3)Gal almost disappears, indicating that the presence of the ␣(1,2)fucosyltransferase (HT) gene/enzyme alters the synthesis of Gal␣(1,3)Gal. A possible mechanism to account for these findings is enzyme location within the Golgi apparatus. We examined the effect of Golgi localization by exchanging the cytoplasmic tails of HT and GT; if Golgi targeting signals are contained within the cytoplasmic tail sequences of these enzymes then a "tail switch" would permit GT first access to the substrate and thereby reverse the observed dominance of HT. Two chimeric glycosyltransferase proteins were constructed and compared with the normal glycosyltransferases after transfection into COS cells. The chimeric enzymes showed K m values and cell surface carbohydrate expression comparable with normal glycosyltransferases. Coexpression of the two chimeric glycosyltransferases resulted in cell surface expression of Gal␣(1,3)Gal, and virtually no HT product was expressed. Thus the cytoplasmic tail of HT determines the temporal order of action, and therefore dominance, of these two enzymes.Glycosylation is a complex form of posttranslational modification of proteins where glycosyltransferases, located in the endoplasmic reticulum and Golgi apparatus, catalyze the sequential transfer of monosaccharides from nucleotide sugars to saccharide acceptors resulting in mature oligosaccharides (1). Thus the large number of both N-and O-linked carbohydrate side chains, consisting of structures of three or 4 simple monosaccharides, can have the different glycosylation patterns generated by the sequential and coordinated action of more than 100 different glycosyltransferases (2). In this process, the product of one transferase serves as the substrate for the next transferase (3), with the differences in the glycosylation pattern being due to variation in the topology and function and the level of expression of the glycosyltransferase within the Golgi complex (3).Evidence to date based on immunoelectron microscopy and cell fractionation studies suggests that the glycosyltransferases are sequentially distributed within the Golgi compartments in approximately the observed order of glycosylation (4). A number of studies using hybrid molecules have examined mechanisms for the sorting and retention of glycosyltransferases within the Golgi and have identified the transmembrane domains of ␣(2,6)sialyltransferase, ␤(1,4)galactosyltransferase, and N-acetylglucosaminyltransferase I as playing a central role in their Golgi localization (5); however, cytoplasmic tail and stem region sequences in ␣(2,6)sialyltransferase and Nacetylglucosaminyltransferase I also contain important structural elements within their cytoplasmic domains that augment the efficiency of Golgi localization of these proteins (5). Thus the sequences that localize glycosyltransferases within di...

Induction of complement attack on human cells by Gal(alpha1,3)Gal xenoantigen expression as a gene therapy approach to cancer

“…15 This was proposed to be because the Gal(␤1,4)GlcNAc-R substrate is common to ␣1,2FT and ␣1,3GT and due to the earlier location of ␣1,2FT within the glycosylation modification pathway in the Golgi. 16 We therefore examined endogenous ␣1,2FT expression by flow cytometry using FITC-conjugated UEA-I lectin to detect Fuc(␣1,2)Gal. Both ECV304 and EAhy926 were positive, while melanoma, TE671 and HT1080 cells were negative (data not shown).…”

Golgi Localization of Carbohydrate Sulfotransferases Is a Determinant of L-selectin Ligand Biosynthesis

“…Several groups have shown that the catalytic domains of glycosyltransferases can be replaced with heterologous proteins, such as GFP, without detriment to proper Golgi localization (41). Likewise, the exchange of localization domains between different glycosyltransferases has yielded active proteins whose location within the Golgi depends on the origin of the localization domain, attesting to the modularity of the protein (35,36,69,70).…”

“…We confirmed this by transient transfection of the GlcNAc6ST-3-EYFP-expressing CHO cells with the gene encoding GlyCAM-1 fused to a human immunoglobulin G heavy chain (GlyCAM-IgG). The secreted molecule was isolated using protein A-Sepharose, and enzyme activity was determined by incorporation of 35 S-labeled sulfate into the glycoprotein. As shown in Fig.…”

“…Indeed, localization of glycosyltransferases to particular Golgi cisternae can dictate biosynthetic pathways by providing access to substrates in a prescribed order (35,36). We considered the possibility that the organization of GlcNAc6STs, specifically GlcNAc6ST-1, -2, and -3, within the Golgi compartment might dictate their biological substrates.…”

The Cytoplasmic Tail of α1,2-Fucosyltransferase Contains a Sequence for Golgi Localization

“…Furthermore, switching the cytoplasmic tails of these transferases altered the dominant effect of FT indicating that the cytoplasmic tail affects the localization sites of the enzymes (17).…”