Background: Glycans are synthesized in the Golgi by sequentially acting glycosyltransferases, but it is not known how their functions are coordinated in live cells. Results: N-and O-glycosyltransferases form enzymatically active homo-and/or heteromeric complexes. Conclusion: Glycosyltransferases function as physically distinct enzyme complexes rather than single enzymes. Significance: The results help understand the overall functioning of the Golgi glycosylation pathways both in health and disease.

One of the mechanisms by which epithelial cells regulate intracellular pH is exchanging bicarbonate for Cl − . We tested the hypothesis that in ameloblasts the anion exchanger-2 (Ae2) is involved in pH regulation during maturation stage amelogenesis. Quantitative X-ray microprobe mineral content analysis, scanning electron microscopy, histology, micro-computed tomography and Ae2 immunolocalisation analyses were applied to Ae2-deficient and wild-type mouse mandibles. Immunolocalisation of Ae2 in wild-type mouse incisors showed a very strong expression of Ae2 in the basolateral membranes of the maturation stage ameloblasts. Strikingly, zones of contiguous ameloblasts were found within the maturation stage in which Ae2 expression was extremely low as opposed to neighbouring cells. Maturation stage ameloblasts of the Ae2 a,b −/− mice failed to stain for Ae2 and showed progressive disorganisation as enamel development advanced. Maturation stage enamel of the Ae2 a,b −/− mice contained substantially less mineral and more protein than wild-type enamel as determined by quantitative X-ray microanalysis. Incisor enamel was more severely affected than molar enamel. Scanning electron microscopy revealed that the rod-inter-rod structures of the Ae2 a,b −/− mice incisor enamel were absent. Mineral content of dentine and bone of Ae2 a,b −/− mice was not significantly different from wild-type mice. The enamel from knockout mouse teeth wore down much faster than that from wild-type litter mates. Basolateral bicarbonate secretion via the anionic exchanger Ae2 is essential for mineral growth in the maturation stage enamel. The observed zonal expression of Ae2 in the maturation stage ameloblasts is in line with a model for cyclic proton secretion during maturation stage amelogenesis.

Abnormal glycosylation of cellular glycoconjugates is a common phenotypic change in many human tumors. Here, we explore the possibility that an altered Golgi pH may also be responsible for these cancer-associated glycosylation abnormalities. We show that a mere dissipation of the acidic Golgi pH results both in increased expression of some cancer-associated carbohydrate antigens and in structural disorganization of the Golgi apparatus in otherwise normally glycosylating cells. pH dependence of these alterations was confirmed by showing that an acidification-defective breast cancer cell line (MCF-7) also displayed a fragmented Golgi apparatus, whereas the Golgi apparatus was structurally normal in its acidification-competent subline (MCF-7/AdrR). Acidification competence was also found to rescue normal glycosylation potential in MCF-7/AdrR cells. Finally, we show that abnormal glycosylation is also accompanied by similar structural disorganization and fragmentation of the Golgi apparatus in colorectal cancer cells in vitro and in vivo. These results suggest that an inappropriate Golgi pH may indeed be responsible for the abnormal Golgi structure and lowered glycosylation potential of the Golgi apparatus in malignant cells. ß

The purpose of this study was to clarify the expression of TMPRSS2 in mice during development and to compare the tissue distribution of the transcripts in adult mouse and human tissues. Mouse TMPRSS2 cDNA was cloned; the predicted amino acid sequence contains 490 residues sharing 81.4% similarity with human TMPRSS2. According to northern blots, mouse TMPRSS2 is expressed mainly in the prostate and kidney, while human TMPRSS2 is expressed in the prostate, colon, stomach, and salivary gland. In situ hybridization analyses of mouse embryos and adult tissues revealed that TMPRSS2 was expressed in the epithelia of the gastrointestinal, urogenital, and respiratory tracts. Expression was very selective and constant after the gene was turned on during development. Expression of TMPRSS2 was localized in the luminal epithelial cells of the mouse and human prostate. The information presented here will be useful in further studies regarding the function and physiological significance of TMPRSS2.

Acidic pH of the Golgi lumen is known to be crucial for correct glycosylation, transport and sorting of proteins and lipids during their transit through the organelle. To better understand why Golgi acidity is important for these processes, we have examined here the most pH sensitive events in N-glycosylation by sequentially raising Golgi luminal pH with chloroquine (CQ), a weak base. We show that only a 0.2 pH unit increase (20 microM CQ) is sufficient to markedly impair terminal alpha(2,3)-sialylation of an N-glycosylated reporter protein (CEA), and to induce selective mislocalization of the corresponding alpha(2,3)-sialyltransferase (ST3) into the endosomal compartments. Much higher pH increase was required to impair alpha(2,6)-sialylation, or the proximal glycosylation steps such as beta(1,4)-galactosylation or acquisition of Endo H resistance, and the steady-state localization of the key enzymes responsible for these modifications (ST6, GalT I, MANII). The overall Golgi morphology also remained unaltered, except when Golgi pH was raised close to neutral. By using transmembrane domain chimeras between the ST6 and ST3, we also show that the luminal domain of the ST6 is mainly responsible for its less pH sensitive localization in the Golgi. Collectively, these results emphasize that moderate Golgi pH alterations such as those detected in cancer cells can impair N-glycosylation by inducing selective mislocalization of only certain Golgi glycosyltransferases.