We recently showed that a class of novel carboxylated N-glycans was constitutively expressed on endothelial cells. Activated, but not resting, neutrophils expressed binding sites for the novel glycans. We also showed that a mAb against these novel glycans (mAbGB3.1) inhibited leukocyte extravasation in a murine model of peritoneal inflammation. To identify molecules that mediated these interactions, we isolated binding proteins from bovine lung by their differential affinity for carboxylated or neutralized glycans. Two leukocyte calcium-binding proteins that bound in a carboxylate-dependent manner were identified as S100A8 and annexin I. An intact N terminus of annexin I and heteromeric assembly of S100A8 with S100A9 (another member of the S100 family) appeared necessary for this interaction. A mAb to S100A9 blocked neutrophil binding to immobilized carboxylated glycans. Purified human S100A8/A9 complex and recombinant human annexin I showed carboxylate-dependent binding to immobilized bovine lung carboxylated glycans and recognized a subset of mannose-labeled endothelial glycoproteins immunoprecipitated by mAbGB3.1. Saturable binding of S100A8/A9 complex to endothelial cells was also blocked by mAbGB3.1. These results suggest that the carboxylated glycans play important roles in leukocyte trafficking by interacting with proteins known to modulate extravasation.

No abstract

PDI1 is the essential gene encoding protein disulfide isomerase in yeast. The Saccharomyces cerevisiae genome, however, contains four other nonessential genes with homology to PDI1: MPD1, MPD2, EUG1, and EPS1. We have investigated the effects of simultaneous deletions of these genes. In several cases, we found that the ability of the PDI1 homologues to restore viability to a pdi1-deleted strain when overexpressed was dependent on the presence of low endogenous levels of one or more of the other homologues. This shows that the homologues are not functionally interchangeable. In fact, Mpd1p was the only homologue capable of carrying out all the essential functions of Pdi1p. Furthermore, the presence of endogenous homologues with a CXXC motif in the thioredoxin-like domain is required for suppression of a pdi1 deletion by EUG1 (which contains two CXXS active site motifs). This underlines the essentiality of protein disulfide isomerase-catalyzed oxidation. Most mutant combinations show defects in carboxypeptidase Y folding as well as in glycan modification. There are, however, no significant effects on ER-associated protein degradation in the various protein disulfide isomerase-deleted strains.

MPI encodes phosphomannose isomerase, which interconverts fructose 6-phosphate and mannose 6-phosphate (Man-6-P), used for glycoconjugate biosynthesis.

No abstract

Bifidobacterium breve Bif195 Protects Against Small-intestinal Damage Caused by Acetylsalicylic Acid in Healthy Volunteers A randomized, placebo-controlled, double-blind clinical trial Aspirin + Bif195 Aspirin + Placebo = 31 Bif195 Placebo = 35 Aspirin Bif195 / Placebo See Covering the Cover synopsis on 587. BACKGROUND & AIMS: Enteropathy and small-intestinal ulcers are common adverse effects of nonsteroidal antiinflammatory drugs such as acetylsalicylic acid (ASA). Safe, cytoprotective strategies are needed to reduce this risk. Specific bifidobacteria might have cytoprotective activities, but little is known about these effects in humans. We used serial video capsule endoscopy (VCE) to assess the efficacy of a specific Bifidobacterium strain in healthy volunteers exposed to ASA. METHODS: We performed a single-site, double-blind, parallelgroup, proof-of-concept analysis of 75 heathy volunteers given ASA (300 mg) daily for 6 weeks, from July 31 through October 24, 2017. The participants were randomly assigned (1:1) to groups given oral capsules of Bifidobacterium breve (Bif195) (!5 Â 10 10 colony-forming units) or placebo daily for 8 weeks. Small-intestinal damage was analyzed by serial VCE at 6 visits. The area under the curve (AUC) for intestinal damage (Lewis score) and the AUC value for ulcers were the primary and first-ranked secondary end points of the trial, respectively. RESULTS: Efficacy data were obtained from 35 participants given Bif195 and 31 given placebo. The AUC for Lewis score was significantly lower in the Bif195 group (3040 ± 1340 arbitrary units) than the placebo group (4351 ± 3195) (P ¼ .0376). The AUC for ulcer number was significantly lower in the Bif195 group (50.4 ± 53.1 arbitrary units) than in the placebo group (75.2 ± 85.3 arbitrary units) (P ¼ .0258). Twelve adverse events were reported from the Bif195 group and 20 from the placebo group. None of the events was determined to be related to Bif195 intake. CONCLUSIONS: In a randomized, double-blind trial of healthy volunteers, we found oral Bif195 to safely reduce the risk of small-intestinal enteropathy caused by ASA. ClinicalTrials.gov no: NCT03228589.

Intestinal biopsy in a boy with gastroenteritis-induced protein-losing enteropathy (PLE) showed loss of heparan sulfate (HS) and syndecan-1 core protein from the basolateral surface of the enterocytes, which improved after PLE subsided. Isoelectric focusing analysis of serum transferrin indicated a congenital disorder of glycosylation (CDG) and subsequent analysis showed three point mutations in the ALG6 gene encoding an ␣1,3-glucosyltransferase needed for the addition of the first glucose to the dolichol-linked oligosaccharide. The maternal mutation, C998T, causing an A333V substitution, has been shown to cause CDG-Ic, whereas the two paternal mutations, T391C (Y131H) and C924A (S308R) have not previously been reported. The mutations were tested for their ability to rescue faulty N-linked glycosylation of carboxypeptidase Y in an ALG6-deficient Saccharomyces cerevisiae strain. Normal human ALG6 rescues glycosylation and A333V partially rescues, whereas the combined paternal mutations (Y131H and S308R) are ineffective. Underglycosylation resulting from each of these mutations is much more severe in rapidly dividing yeast. Similarly, incomplete protein glycosylation in the patient is most severe in rapidly dividing enterocytes during gastroenteritis-induced stress. 1-4 These defects reduce the amount or change the structure of the lipid-linked oligosaccharide (LLO) precursor for N-linked glycosylation, leading to underglycosylation of many proteins, 3 including antithrombin-III, factor XI, or protein C. This reduces their levels, leaving patients at risk for coagulopathy. Mutations in phosphomannose isomerase (MPI, causing CDG-Ib) and phosphomannomutase (PMM2, causing CDG-Ia) reduce the amount of GDP-Man, the immediate precursor of LLO,5 thus reducing the amount of N-linked glycosylation. Mutations in ALG6, which encodes an ␣1,3-glucosyltransferase, cause CDG-Ic. 6 -8 This enzyme is required for the addition of the first of three glucose residues to LLO, and without the first glucose, further glucosylation is prevented. The nonglucosylated precursor oligosaccharide is a poor substrate for the oligosaccharyltransferase complex and is inefficiently transferred to proteins. 9,10

The sorting of the yeast proteases proteinase A and carboxypeptidase Y to the vacuole is a saturable, receptor-mediated process. Information sufficient for vacuolar sorting of the normally secreted protein invertase has in fusion constructs previously been found to reside in the propeptide of proteinase A. We found that sorting of such a hybrid protein is dependent on the vacuolar protein-sorting receptor Vps10p. This was unexpected, as strains disrupted for VPS10 sort more than 85% of the proteinase A to the vacuole. Consistent with a role for Vps10p in sorting of proteinase A, we found that 1) overproduction of Vps10p suppressed the missorting phenotype associated with overproduction of proteinase A, 2) overproduction of proteinase A induced missorting of carboxypeptidase Y, 3) vacuolar sorting of proteinase A in a ⌬vps10 strain was readily saturated by modest overproduction of proteinase A, and 4) Vps10p and proteinase A interact directly and specifically as shown by chemical cross-linking. Interestingly, overexpression of two telomere-linked VPS10 homologues, VTH1 and VTH2 suppressed the missorting phenotypes of a ⌬vps10 strain. However, disruption of the VTH1 and VTH2 genes did not affect the sorting of proteinase A. We conclude that proteinase A utilizes at least two mechanisms for sorting, a Vps10p-dependent path and a Vth1p/ Vth2p/Vps10p-independent path.The yeast vacuole contains a number of soluble hydrolases that are delivered to this organelle via the endoplasmic reticulum and the Golgi complex. Thus, both vacuolar and secretory proteins transit through these early compartments of the secretory pathway on their way to their final destination. Sorting takes place in a late subcompartment of the Golgi complex, the trans Golgi network, where soluble vacuolar proteins are diverted to the late endosome by an active, saturable mechanism (1-4). Carboxypeptidase Y (CPY) 1 and proteinase A (PrA) have been the model enzymes in most studies of the biosynthesis of soluble yeast vacuolar proteins. These enzymes are synthesized as precursor forms that upon arrival in the vacuole are activated by proteolytic removal of an N-terminal propeptide. The vacuolar sorting signal of CPY is located in the propeptide (5-8), and the VPS10 gene encodes the receptor (Vps10p), which interacts with this signal and is responsible for the sorting of CPY (9). Disruption of VPS10 results in complete mislocalization of CPY but does not strongly affect vacuolar sorting of PrA, indicating that PrA can be sorted to the vacuole by an alternate mechanism (9). The 54-amino acid propeptide of PrA can direct the normally periplasmic enzyme invertase to the vacuole, indicating that it contains sorting information (10). More precise identification of this propeptide-located sorting signal turned out to be difficult, as the propeptide proved to be essential for folding, and thus also endoplasmic reticulum exit, of PrA (11). In a previous study, the role of the PrA propeptide in folding of the enzyme was investigated by random substitution of eit...