The core fucosylation (␣1,6-fucosylation) of glycoproteins is widely distributed in mammalian tissues, and is altered under pathological conditions. To investigate physiological functions of the core fucose, we generated ␣1,6-fucosyltransferase (Fut8)-null mice and found that disruption of Fut8 induces severe growth retardation and death during postnatal development. Histopathological analysis revealed that Fut8 ؊/؊ mice showed emphysema-like changes in the lung, verified by a physiological compliance analysis. Biochemical studies indicated that lungs from Fut8 ؊/؊ mice exhibit a marked overexpression of matrix metalloproteinases (MMPs), such as MMP-12 and MMP-13, highly associated with lung-destructive phenotypes, and a down-regulation of extracellular matrix (ECM) proteins such as elastin, as well as retarded alveolar epithelia cell differentiation. These changes should be consistent with a deficiency in TGF-␤1 signaling, a pleiotropic factor that controls ECM homeostasis by down-regulating MMP expression and inducing ECM protein components. In fact, Fut8 ؊/؊ mice have a marked dysregulation of TGF-␤1 receptor activation and signaling, as assessed by TGF-␤1 binding assays and Smad2 phosphorylation analysis. We also show that these TGF-␤1 receptor defects found in Fut8 ؊/؊ cells can be rescued by reintroducing Fut8 into Fut8 ؊/؊ cells. Furthermore, exogenous TGF-␤1 potentially rescued emphysema-like phenotype and concomitantly reduced MMP expression in Fut8 ؊/؊ lung. We propose that the lack of core fucosylation of TGF-␤1 receptors is crucial for a developmental and progressive͞ destructive emphysema, suggesting that perturbation of this function could underlie certain cases of human emphysema.fucosylation ͉ glycobiology ͉ matrix metalloproteinase

Changes in oligosaccharide structures have been reported in certain types of malignant transformations and, thus, could be used for tumor markers in certain types of cancer. In the case of pancreatic cancer cell lines, a variety of fucosylated proteins are secreted into their conditioned media. To identify fucosylated proteins in the serum of patients with pancreatic cancer, we performed western blot analyses using Aleuria Aurantica Lectin (AAL), which is specific for fucosylated structures. An 40 kD protein was found to be highly fucosylated in pancreatic cancer and an N-terminal analysis revealed that it was the b chain of haptoglobin. While the appearance of fucosylated haptoglobin has been reported in other diseases such as hepatocellular carcinoma, liver cirrhosis, gastric cancer and colon cancer, the incidence was significantly higher in the case of pancreatic cancer. Fucosylated haptoglobin was observed more frequently at the advanced stage of pancreatic cancer and disappeared after an operation. A mass spectrometry analysis of haptoglobin purified from the serum of patients with pancreatic cancer and the medium from a pancreatic cancer cell line, PSN-1, showed that the a 1-3/a 1-4/a 1-6 fucosylation of haptoglobin was increased in pancreatic cancer. When a hepatoma cell line, Hep3B, was cultured with the conditioned media from pancreatic cancer cells, haptoglobin secretion was dramatically increased. These findings suggest that fucosylated haptoglobin could serve as a novel marker for pancreatic cancer. Two possibilities were considered in terms of the fucosylation of haptoglobin. One is that pancreatic cancer cells, themselves, produce fucosylated haptoglobin; the other is that pancreatic cancer produces a factor, which induces the production of fucosylated haptoglobin in the liver. ' 2005 Wiley-Liss, Inc.Key words: haptoglobin; pancreatic cancer; fucosylation; tumor marker; mass spectrometry; oligosaccharide; lectin; fucosyltransferase Pancreatic cancer is currently one of the leading causes of cancer-related deaths and the overall 5-year survival has been reported to be less than 5%.

It was found in our previous studies that the concentration of fucosylated haptoglobin had increased in the sera of patients with pancreatic cancer (PC) compared to those of other types of cancer and normal controls. Haptoglobin, an acute phase protein, has four potential N-glycosylation sites, although it remains unknown which site is responsible for the change in fucosylated N-glycans. In the present study, site-specific N-glycan structures of haptoglobin in sera obtained from patients with PC or chronic pancreatitis (CP) were analyzed using liquid chromatography-electrospray ionization mass spectrometry. Mass spectrometry analyses demonstrated that concentrations of total fucosylated di-, tri-and tetra-branched glycans of haptoglobin increased in the sera of PC patients. Tri-antennary N-glycans containing a Lewis X-type fucose markedly increased at the Asn211 site of haptoglobin Nglycans. While fucosylated N-glycans derived from serum haptoglobin of patients with CP slightly increased, di-fucosylated tetraantennary N-glycans were observed only at this site in PC patients, and were absent in the haptoglobin of normal controls and individuals with CP. Thus, the present study provides evidence that site-specific analyses of N-glycans may be useful as novel tumor markers for PC.

Copper-zinc superoxide dismutase (SOD1) plays a protective role against oxidative stress. On the other hand, recent studies suggest that SOD1 itself is a major target of oxidative damage and has its own pathogenicity in various neurodegenerative diseases, including familial amyotrophic lateral sclerosis. Only human and great ape SOD1s among mammals have the highly reactive free cysteine residue, Cys 111 , at the surface of the SOD1 molecule. The purpose of this study was to investigate the role of Cys 111 in the oxidative damage of the SOD1 protein, by comparing the oxidative susceptibility of recombinant human SOD1 modified with 2-mercaptoethanol at Cys 111 (2-ME-SOD1) to wild-type SOD1. Wild-type SOD1 was more sensitive to oxidation by hydrogen peroxide-generating fragments, oligomers, and charge isomers compared with 2-ME-SOD1. Moreover, wild-type SOD1, but not 2-ME-SOD1, generated an upper shifted band in reducing SDS-PAGE even by air oxidation. Using mass spectrometry and limited proteolysis, this upper band was identified as an oxidized subunit of SOD1; the sulfhydryl group (Cys-SH) of Cys 111 was selectively oxidized to cysteine sulfinic acid (Cys-SO 2 H) and to cysteine sulfonic acid (Cys-SO 3 H). (2). Moreover, incubation with excess H 2 O 2 caused oxidation of almost all histidine and cysteine residues (3), fragmentation (4, 5) and aggregation (6, 7) of SOD1 itself. Co-incubation with bicarbonate and H 2 O 2 also induced bicarbonate radical anion formation, resulting in oligomerization of human SOD1 (8).The familial form of amyotrophic lateral sclerosis (ALS) is associated with specific mutations in the SOD1 gene (SOD1) that encodes 153 amino acids (9, 10). To date, more than 110 familial ALS (FALS)-causing mutations in SOD1 have been identified (available on the World Wide Web); however, the mechanism by which SOD1 mutants induce ALS remains unknown. The presence of intracellular aggregates that contain SOD1 in spinal cord motor neurons is thought to be a pathological hallmark of ALS. In particular, FALS-linked mutant SOD1s are prone to misfolding and aggregation (11,12). Recently, Ezzi et al. (7) reported that even wild-type SOD1 results in aggregation after oxidation, and the oxidized wildtype SOD1 gains properties like FALS mutant SOD1s. In addition to ALS, oxidative damaged SOD1 proteins were detected in the brains of patients with Alzheimer and Parkinson diseases (13). These findings suggest that oxidized SOD1 plays a role in the pathophysiology of various neurodegenerative diseases.

The β-site amyloid precursor protein cleaving enzyme-1 (BACE1), an essential protease for the generation of amyloid-β (Aβ) peptide, is a major drug target for Alzheimer's disease (AD). However, there is a concern that inhibiting BACE1 could also affect several physiological functions. Here, we show that BACE1 is modified with bisecting N-acetylglucosamine (GlcNAc), a sugar modification highly expressed in brain, and demonstrate that AD patients have higher levels of bisecting GlcNAc on BACE1. Analysis of knockout mice lacking the biosynthetic enzyme for bisecting GlcNAc, GnT-III (Mgat3), revealed that cleavage of Aβ-precursor protein (APP) by BACE1 is reduced in these mice, resulting in a decrease in Aβ plaques and improved cognitive function. The lack of this modification directs BACE1 to late endosomes/lysosomes where it is less colocalized with APP, leading to accelerated lysosomal degradation. Notably, other BACE1 substrates, CHL1 and contactin-2, are normally cleaved in GnT-III-deficient mice, suggesting that the effect of bisecting GlcNAc on BACE1 is selective to APP. Considering that GnT-III-deficient mice remain healthy, GnT-III may be a novel and promising drug target for AD therapeutics.