Glycosylation Changes Triggered by the Differentiation of Monocytic THP-1 Cell Line into Macrophages

Proteomic

Glycolipid-dependent sorting of melanosomal from lysosomal membrane proteins by lumenal determinants

Melanosomes are lysosome-related organelles that coexist with lysosomes in mammalian pigment cells. Melanosomal and lysosomal membrane proteins share similar sorting signals in their cytoplasmic tail, raising the question how they are segregated. We show that in control melanocytes, the melanosomal enzymes tyrosinase-related protein 1 (Tyrp1) and tyrosinase follow an intracellular Golgi to melanosome pathway, whereas in the absence of glycosphingolipids, they are observed to pass over the cell surface. Unexpectedly, the lysosome-associated membrane protein 1 (LAMP-1) and 2 behaved exactly opposite: they were found to travel through the cell surface in control melanocytes but followed an intracellular pathway in the absence of glycosphingolipids. Chimeric proteins having the cytoplasmic tail of Tyrp1 or tyrosinase were transported like lysosomal proteins, whereas a LAMP-1 construct containing the lumenal domain of Tyrp1 localized to melanosomes. In conclusion, the lumenal domain contains sorting information that guides Tyrp1 and probably tyrosinase to melanosomes by an intracellular route that excludes lysosomal proteins and requires glucosylceramide

Hyperacidification of trans-Golgi network and endo/lysosomes in melanocytes by glucosylceramide-dependent V-ATPase activity

Sphingolipids are considered to play a key role in protein sorting and membrane trafficking. In melanocytic cells, sorting of lysosomal and melanosomal proteins requires the sphingolipid glucosylceramide (GlcCer). This sorting information is located in the lumenal domain of melanosomal proteins. We found that two processes dependent on lumenal pH, protein sialylation and lysosomal acid lipase (LAL) activity were aberrant in GM95 melanocyte cells, which do not produce glycosphingolipids. Using fluorescence lifetime imaging microscopy (FLIM), we found that the lumenal pH in the trans-Golgi network and lysosomes of wild-type melanocyte MEB4 cells are >1 pH unit lower than GM95 cells and fibroblasts. In addition to the lower pH found in vivo, the in vitro activity of the proton pump, the vacuolar-type H+-translocating ATPase (V-ATPase), was twofold higher in MEB4 compared to GM95 cells. The apparent Ki for inhibition of the V-ATPase by concanamycin A and archazolid A, which share a common binding site on the c-ring, was lower in glycosphingolipid-deficient GM95 cells. No difference between the MEB4 and GM95 cells was found for the V-ATPase inhibitors apicularen A and salicylihalimide. We conclude that hyperacidification in MEB4 cells requires glycosphingolipids and propose that low pH is necessary for protein sorting and melanosome biogenesis. Furthermore, we suggest that glycosphingolipids are indirectly involved in protein sorting and melanosome biogenesis by stimulating the proton pump, possibly through binding of GlcCer. These experiments establish, for the first time, a link between pH, glycosphingolipids and melanosome biogenesis in melanocytic MEB4 cells, to suggest a role for glycosphingolipids in hyperacidification in melanocytes

B4GALNT2 gene expression controls the biosynthesis of Sd^a and sialyl Lewis X antigens in healthy and cancer human gastrointestinal tract.

The histo blood group carbohydrate Sda antigen and its cognate biosynthetic enzyme B4GALNT2 show the highest level of expression in normal colon. Their dramatic down regulation previously observed in colon cancer tissues could play a role in the concomitant elevation of the selectin ligand sLex, involved in metastasis. However, down regulation of sLe x expression by B4GALNT2 has been so far demonstrated in vitro, but not in tissues. The human B4GALNT2 gene specifies at least two transcripts, diverging in the first exon, never studied in normal and cancer tissues. The long form contains a 253 nt exon 1L; the short form contains a 38 nt exon 1S. Using qPCR, we showed that cell lines and normal or cancerous colon, expressed almost exclusively the short form, while the long form was mainly expressed by the embryonic colon fibroblast cell line CCD112CoN. Immunochemistry approaches using colon cancer cells permanently expressing either B4GALNT2 cDNAs as controls, led to the observation of several protein isoforms in human normal and cancerous colon, and cell lines. We showed that tissues expressing B4GALNT2 protein isoforms were able to induce Sda and to inhibit sLe x expression; both of which are expressed mainly on PNGase F-insensitive carbohydrate chains. Concomitant expression of B4GALNT2 and siRNA-mediated inhibition of FUT6, the major fucosyltransferase involved in sLex synthesis in colon, resulted in a cumulative inhibition of sLex. In normal colon samples a significant relationship between sLex expression and the ratio between FUT6/B4GALNT2 activities exists, demonstrating for the first time a role for B4GALNT2 in sLex inhibition in vivo

The extended cytoplasmic tail of the human B4GALNT2 is critical for its Golgi targeting and post-Golgi sorting

The Sd(a)/Cad antigen reported on glycoconjugates of human tissues has an increasingly recognized wide impact on the physio-pathology of different biological systems. The last step of its biosynthesis relies on the enzymatic activity of the 1,4-N-acetylgalactosaminyltransferase-II (B4GALNT2), which shows the highest expression level in healthy colon. Previous studies reported the occurrence in human colonic cells of two B4GALNT2 protein isoforms that differ in the length of their cytoplasmic tail, the long isoform showing an extended 66-amino acid tail. We examined here, the subcellular distribution of the two B4GALNT2 protein isoforms in stably transfected colonic LS174T cells and in transiently transfected HeLa cells using fluorescence microscopy. While a similar subcellular distribution at the trans-Golgi cisternae level was observed for the two isoforms, our study pointed to an atypical subcellular localization of the long B4GALNT2 isoform into dynamic vesicles. We demonstrated a critical role of its extended cytoplasmic tail for its Golgi targeting and post-Golgi sorting and highlighted the existence of a newly described post-Golgi sorting signal as well as a previously undescribed fate of a Golgi glycosyltransferase.DatabaseThe proteins 1,4GalNAcT II, 1,4-GalT1, FucT I, FucT VI and ST3Gal IV are noted B4GALNT2, B4GALT1, FUT1, FUT6 and ST3GAL4, whereas the corresponding human genes are noted B4GALNT2, B4GALT1, FUT1, FUT6 and ST3GAL4 according to the HUGO nomenclature