Proteins internalized into the endocytic pathway are usually degraded. Efficient proteolysis requires denaturation, induced by acidic conditions within lysosomes, and reduction of inter-and intrachain disulfide bonds. Cytosolic reduction is mediated enzymatically by thioredoxin, but the mechanism of lysosomal reduction is unknown. We describe here a lysosomal thiol reductase optimally active at low pH and capable of catalyzing disulfide bond reduction both in vivo and in vitro. The active site, determined by mutagenesis, consists of a pair of cysteine residues separated by two amino acids, similar to other enzymes of the thioredoxin family. The enzyme is a soluble glycoprotein that is synthesized as a precursor. After delivery into the endosomal͞lysosomal system by the mannose 6-phosphate receptor, N-and C-terminal prosequences are removed. The enzyme is expressed constitutively in antigen-presenting cells and induced by IFN-␥ in other cell types, suggesting a potentially important role in antigen processing. R eduction, oxidation, and isomerization of protein disulfide bonds in the cytosol and endoplasmic reticulum (ER) of eukaryotic cells are carried out by enzymes of the thioredoxin family (1). Protein disulfide isomerase and related molecules catalyze the formation and isomerization of protein disulfide bonds in the ER (2-4). Thioredoxin and glutaredoxin catalyze reduction of disulfide bonds in the cytosol and nucleus (1, 5). These enzymes use oxidized cofactors (e.g., oxidized glutathione) as electron sinks or reduced cofactors (e.g., glutathione) as electron donors for oxidation or reduction of protein disulfide bonds, respectively (5, 6). Members of the thioredoxin family often share little sequence similarity but do possess a common active site (WCGH͞PCK) and folding pattern (7-10). The cysteine residues in the active site are believed to act by transferring electrons between themselves and either the substrate protein or cofactors (9).Disulfide bond reduction also occurs in the endocytic pathway. Most proteins that enter the endocytic pathway are degraded in lysosomes to small peptides and free amino acids. Denaturation of proteins is a prerequisite for lysosomal proteolysis (reviewed in ref . 11) and is facilitated by the acidic pH of the lysosome (12). A reducing environment within the endocytic pathway also facilitates denaturation by cleaving disulfide bonds in substrate proteins (reviewed in ref. 13). Various groups have demonstrated reducing activity in lysosomes (14-17) and endosomes (18). Although the presence of excess cysteine favors the reduction of disulfide bonds (16), the process is not favored at low pH (19), and no enzyme(s) that catalyzes reduction in these compartments has been described. We have now defined such an enzyme and have named it Gamma interferon-inducible lysosomal thiol-reductase (GILT). GILT was originally described by Luster et al. (20) and called IP30. It is synthesized as a 224-aa precursor that is transported to endocytic compartments by mannose-6-phosphate receptors...

Processing of proteins for major histocompatibility complex (MHC) class II-restricted presentation to CD4-positive T lymphocytes occurs after they are internalized by antigen-presenting cells (APCs). Antigenic proteins frequently contain disulfide bonds, and their reduction in the endocytic pathway facilitates processing. In humans, a gamma interferon-inducible lysosomal thiol reductase (GILT) is constitutively present in late endocytic compartments of APCs. Here, we identified the mouse homolog of GILT and generated a GILT knockout mouse. GILT facilitated the processing and presentation to antigen-specific T cells of protein antigens containing disulfide bonds. The response to hen egg lysozyme, a model antigen with a compact structure containing four disulfide bonds, was examined in detail.

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Long-lasting tumor immunity requires functional mobilization of CD8+ and CD4+ T lymphocytes. CD4+ T cell activation is enhanced by presentation of shed tumor antigens by professional antigen-presenting cells (APCs), coupled with display of similar antigenic epitopes by major histocompatibility complex class II on malignant cells. APCs readily processed and presented several self-antigens, yet T cell responses to these proteins were absent or reduced in the context of class II+ melanomas. T cell recognition of select exogenous and endogenous epitopes was dependent on tumor cell expression of γ-interferon–inducible lysosomal thiol reductase (GILT). The absence of GILT in melanomas altered antigen processing and the hierarchy of immunodominant epitope presentation. Mass spectral analysis also revealed GILT's ability to reduce cysteinylated epitopes. Such disparities in the profile of antigenic epitopes displayed by tumors and bystander APCs may contribute to tumor cell survival in the face of immunological defenses.

We recently identified a gamma-interferon-inducible lysosomal thiol reductase (GILT), constitutively expressed in antigen-presenting cells, that catalyzes disulfide bond reduction both in vitro and in vivo and is optimally active at acidic pH. GILT is synthesized as a 35-kDa precursor, and following delivery to major histocompatibility complex (MHC) class II-containing compartments (MIICs), is processed to the mature 30-kDa form via cleavage of N-and C-terminal propeptides. The generation of MHC class II epitopes requires both protein denaturation and reduction of intra-and interchain disulfide bonds prior to proteolysis. GILT may be important in disulfide bond reduction of proteins delivered to MIICs and consequently in antigen processing. In this report we show that, like its mature form, precursor GILT reduces disulfide bonds with an acidic pH optimum, suggesting that it may also be involved in disulfide bond reduction in the endocytic pathway. We also show that processing of precursor GILT can be mediated by multiple lysosomal proteases and provide evidence that the mechanism of action of GILT resembles that of other thiol oxidoreductases.

Crystal structures of the lectin and epidermal growth factor (EGF)-like domains of P-selectin show 'bent' and 'extended' conformations. An extended conformation would be 'favored' by forces exerted on a selectin bound at one end to a ligand and at the other end to a cell experiencing hydrodynamic drag forces. To determine whether the extended conformation has higher affinity for ligand, we introduced an N-glycosylation site to 'wedge open' the interface between the lectin and EGF-like domains of P-selectin. This alteration increased the affinity of P-selectin for its ligand Pselectin glycoprotein 1 (PSGL-1) and thereby the strength of P-selectin-mediated rolling adhesion. Similarly, an asparagine-to-glycine substitution in the lectin-EGF-like domain interface of L-selectin enhanced rolling adhesion under shear flow. Our results demonstrate that force, by 'favoring' an extended selectin conformation, can strengthen selectin-ligand bonds.Leukocyte migration to inflamed tissues and lymphocyte homing to peripheral lymphoid organs involves a multistep process 1-6 . In the first step, leukocytes tether and roll along vascular endothelia. Rolling exposes leukocytes to chemokines that activate integrins, which arrest leukocyte rolling and mediate leukocyte migration across the endothelia.Rolling results from the hydrodynamic drag force acting on adherent cells. For rolling to be stable, the formation of new receptor-ligand bonds downstream must balance the dissociation of bonds upstream. Leukocyte tethering and rolling are mediated mainly by E-, L-and Pselectins. By initiating transient, rapidly reversible receptor-ligand interactions, these C-type lectin molecules allow a 'zone of adhesion' to move along a vessel wall 7-9 . In contrast, antibody-antigen interactions are unable to support stable rolling over a wide range of shear stresses 10 .Two features contribute to the ability of selectins to mediate stable leukocyte rolling. First, force increases the number of bonds that form between a rolling cell and its substrate, which effectively compensates for the shortening of receptor-ligand bond lifetimes that occurs as force on the bond increases 11 . Cell deformation and tether extension also promote leukocyte rolling 11-13 . Second, individual selectin-ligand bonds are more resistant to force than any other measured receptor-ligand bond 8,11,14 . As force on the receptor-ligand bond increases, the 'off rate' (k off ) increases less for selectin-ligand bonds than for integrin-ligand orCorrespondence should be addressed to T.A.S. (springeroffice@cbr.med.harvard.edu).. Note: Supplementary information is available on the Nature Immunology website. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. Selectins contain an N-terminal calcium-dependent lectin domain, an epidermal growth factor (EGF)-like domain, a variable number of short consensus repeats (SCRs), and transmembrane and cytoplasmic domains 6,18-20 . Selectin ligands consist mainly of mucin-like sialoglycopro...

Integrins are cell membrane proteins that integrate the extracellular and intracellular compartments by binding to ligands on other cells or on the extracellular matrix. Integrins contain two noncovalently associated, transmembrane glycoprotein ␣ and ␤ subunits. A globular headpiece binds ligand, and two long leg regions connect the ligand binding headpiece to the transmembrane and C-terminal cytoplasmic domains. Half of integrin ␣ subunits, including ␣ L , contain a domain of ϳ200 amino acids, known as an inserted (I) or von Willebrand factor A domain that contains the major ligand binding site. Integrin ␣ L ␤ 2 , also known as leukocyte function-associated antigen-1 (LFA-1), 4 is expressed on leukocytes and participates in leukocyte trafficking in inflammation, lymphocyte homing, and T lymphocyte interactions with antigen-presenting cells in immune reactions.The conformational state of the I domain in the ␣ L subunit strongly influences its affinity for ligand. A downward movement of the C-terminal ␣-helix of the I domain, linked to conformational rearrangements at the metal ion-dependent adhesion site that constitutes the binding site, is seen in the ICAM-1-bound structure of the ␣ L I domain (1). This conformation is termed open and has been engineered by locking the loop between the C-terminal ␤-strand and ␣-helix into the open conformation using a disulfide bond. The open I domain, both in isolation or in the context of intact ␣ L ␤ 2 , can support firm adhesion to ICAM-1 that is comparable with activated, wild-type ␣ L ␤ 2 (2-4). Intermediate affinity I domain mutants were also engineered by mutationally introducing disulfide bonds between the C-terminal ␣-helix and other portions of the I domain, resulting in affinities for ICAM-1 between 3 and 9.4 M ( Table 1) Besides the ␣ I domain, integrin ectodomains contain four other domains in the ␣ subunit and eight domains in the ␤ subunit. In addition to conformational change in the I domain, integrins undergo large changes in the overall shape of the ectodomain. On physiological cell surfaces, integrins that lack I domains have been shown to assume multiple conformations with distinct affinity states, with the bent conformation in which the head folds over the legs because of a bend at the knees, being the predominant conformation for integrins in the resting, low affinity state (5). Physiological signals that impinge on integrin cytoplasmic domains, as well as certain activating mAbs, alter the equilibrium between different conformational states and trigger extension. For integrins that lack I domains, two extended conformations that differ in the conformation of their heads and in affinity for ligand have been visualized by electron microscopy and in crystal structures (5). On the cell surface, I domains may exist in the three conformations termed closed, intermediate, and open, as seen in crystal structures. Indeed, we have recently demonstrated that ␣ L ␤ 2 can mediate adhesive interactions even when high affinity ligand binding cannot be detected (6). T...

APRIL (A proliferation-inducing ligand) is a TNF family member that binds two TNF receptor family members, TACI and BCMA. It shares these receptors with the closely related TNF family member, B-cell activating factor (BAFF). Contrary to BAFF, APRIL binds heparan sulfate proteoglycans (HSPGs), which regulates cross-linking of APRIL and efficient signaling. APRIL was originally identified as a growth promoter of solid tumors, and more recent evidence defines APRIL also as an important survival factor in several human B-cell malignancies, such as chronic lymphocytic leukemia (CLL). To target APRIL therapeutically, we developed two anti–human APRIL antibodies (hAPRIL.01A and hAPRIL.03A) that block APRIL binding to BCMA and TACI. Their antagonistic properties are unique when compared with a series of commercially available monoclonal anti–human APRIL antibodies as they prevent in vitro proliferation and IgA production of APRIL-reactive B cells. In addition, they effectively impair the CLL-like phenotype of aging APRIL transgenic mice and, more importantly, block APRIL binding to human B-cell lymphomas and prevent the survival effect induced by APRIL. We therefore conclude that these antibodies have potential for further development as therapeutics to target APRIL-dependent survival in B-cell malignancies.