An engineered protein prevents aggregation of the Aβ peptide and facilitates clearance of Aβ from the brain in a fruit fly model of Alzheimer's disease.

Polarized secretion is crucial in many tissues. The conserved protein modification, O-glycosylation, plays a role in regulating secretion. However, the mechanisms by which this occurs are unknown. Here, we demonstrate that an O-glycosyltransferase functions as a novel regulator of secretion and secretory vesicle formation in vivo by glycosylating the essential Golgi/endoplasmic reticulum protein, Tango1 (Transport and Golgi organization 1), and conferring protection from furin-mediated proteolysis. Loss of the O-glycosyltransferase PGANT4 resulted in Tango1 cleavage, loss of secretory granules, and disrupted apical secretion. The secretory defects seen upon loss of pgant4 could be rescued either by overexpression of Tango1 or by knockdown of a specific furin (Dfur2) in vivo. Our studies elucidate a novel regulatory mechanism whereby secretion is influenced by the yin/yang of O-glycosylation and proteolytic cleavage. Moreover, our data have broader implications for the potential treatment of diseases resulting from the loss of O-glycosylation by modulating the activity of specific proteases.R egulation of secretory vesicle formation and polarized secretion in vivo is crucial to ensure the proper deposition of signaling molecules, morphogens, and matrix components that mediate growth and differentiation. Polarized secretion is also required in many differentiated tissues, such as the digestive tract, where secreted components along the apical surface form the mucous membrane that confers protection from mechanical and microbial insults (1) and provides immunoregulatory signals (2). Indeed, disruptions in the secreted mucous membrane are associated with diseases of the digestive tract, such as colitis and colon cancer (3-6).Recent studies aimed at identifying the factors that influence secretion have elucidated novel proteins that function in unique aspects of secretion, including the enzymes responsible for the addition of sugars to mucins and other proteins (mucin-type O-glycosylation) (7). O-glycosylation is an essential, evolutionarily conserved protein modification (8, 9) that has direct medical relevance, as aberrations are responsible for the human diseases familial tumoral calcinosis (10, 11) and Tn syndrome (12). Loss of this protein modification affected constitutive secretion and Golgi apparatus structure in Drosophila cell culture (7, 13) and secretion in the developing respiratory system in vivo (14). Additionally, loss of O-glycosylation also disrupted secretion of an extracellular matrix protein (Tiggrin) in the developing wing, resulting in aberrant basement membrane formation and disrupted integrin-mediated cell adhesion (15). Mammalian studies have confirmed the effects of O-glycosylation on secretion, as loss of a glycosyltransferase (ppGalNAcT-1) disrupted secretion of laminin and collagen during mammalian organogenesis, altering the composition of the basement membrane and disrupting proper FGF signaling and organ growth (16). Although these studies all point to a role for O-glycosylation in se...

MUC1 is a mucin glycoprotein containing multiple tandem repeats of 20 amino acids, with five serines and threonines that can be O-glycosylated. Here, we investigated the O-glycosylation site occupancy in MUC1 glycoproteins produced in two mutant CHO cell lines, Lec3.2.8.1 and ldlD. We found that the average site occupancy was higher in MUC1 from Lec3.2.8.1 than from ldlD and that the occupancy increased with the number of tandem repeats in the protein and also depended on the culture conditions used for production. Moreover, we describe the successful use of electron-capture dissociation (ECD) fragmentation, coupled to online liquid chromatography mass spectrometry, to determine the glycosylation of individual sites in recombinant MUC1 proteins with 16 tandem repeats. We analyzed MUC1 tandem repeat peptides with 1-5 GalNAc residues by ECD fragmentation and found that the first site to be glycosylated was either Ser-5 or Thr-6, with the addition of a second GalNAc at Thr-14. For peptides with three GalNAc residues, several different variants of glycopeptides were found, indicating a heterogeneous order of glycosylation at this stage. In contrast, only one variant was found for peptides with four GalNAc residues, where Thr-19 in the PDTR motif was left unglycosylated, indicating that this site is glycosylated last. The results gave novel insight into the order of GalNAc substitution in MUC1 in vivo.

Sensitive and fast methods for the profiling of biologically important molecules are highly demanded. Mucins are densely O-glycosylated glycoproteins found at mucosal surfaces and are of great medical interest. Here we describe sensitive methods for the analysis of O-glycans from mucins using gel electrophoresis, and chromatography by nanoLC on graphite columns and structural analysis by electrospray mass spectrometry on a linear trap mass spectrometer.

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