Tyrosinase is a type I membrane protein regulating the pigmentation process in humans. Mutations of the human tyrosinase gene cause the tyrosinase negative type I oculocutaneous albinism (OCAI). Some OCAI mutations were shown to delete the transmembrane domain or to affect its hydrophobic properties, resulting in soluble tyrosinase mutants that are retained in the endoplasmic reticulum (ER). To understand the specific mechanisms involved in the ER retention of soluble tyrosinase, we have constructed a tyrosinase mutant truncated at its C-terminal end and investigated its maturation process. The mutant is retained in the ER, and it is degraded through the proteasomal pathway. We determined that the mannose trimming is required for an efficient degradation process. Moreover, this soluble ER-associated degradation substrate is stopped at the ER quality control checkpoint with no requirements for an ER-Golgi recycling pathway. Co-immmunoprecipitation experiments showed that soluble tyrosinase interacts with calreticulin and BiP/GRP78 (and not calnexin) during its ER transit. Expression of soluble tyrosinase in calreticulin-deficient cells resulted in the export of soluble tyrosinase of the ER, indicating the calreticulin role in ER retention. Taken together, these data show that OCAI soluble tyrosinase is an ER-associated degradation substrate that, unlike other albino tyrosinases, associates with calreticulin and BiP/GRP78. The lack of specificity for calnexin interaction reveals a novel role for calreticulin in OCAI albinism.Tyrosinase (monophenol, dihydroxy-phenylalanine: oxygen oxidoreductase; EC 1.14.18.1) is the rate-limiting enzyme involved in melanin biosynthesis (1, 2). This protein, consisting of a large catalytic lumenal domain that is anchored to the membrane by a C-terminal transmembrane domain and a short cytosolic tail (3), undergoes glycosylation prior to being subjected to the endoplasmic reticulum quality control (4). Although misfolded chains are sorted for the endoplasmic reticulum (ER) 1 -associated degradation (ERAD) pathway, folded tyrosinase is exported out of the ER through the secretory pathway targeting the pigmentation site organelle, the melanosome (5, 6). Mutations in the tyrosinase gene result in the absence of pigmentation and are responsible for oculocutaneous albinism type I (OCAI) in humans (7). OCAI was proposed to be an ER retention disease in which misfolded tyrosinase mutants are retained in the ER by the quality control (8, 9). Calnexin and calreticulin, as components of the quality control, were shown to interact transiently with the monoglucosylated N-glycans of the misfolded polypeptides (10, 11). These lectin chaperones engage the chains into the de-glucosylation/re-glucosylation cycle catalyzed by glucosidase II and glucosyltransferase, with the latter recognizing only incompletely, folded chains. Although the correctly folded polypeptides leave the cycle and the ER, the incompletely folded ones are re-glucosylated and retrieved by calnexin/calreticulin (12, 13). Both lec...

Human CD1d molecules consist of a transmembrane CD1 (cluster of differentiation 1) heavy chain in association with ␤ 2 -microglobulin (␤ 2 m). Assembly occurs in the endoplasmic reticulum (ER) and involves the initial glycan-dependent association of the free heavy chain with calreticulin and calnexin and the thiol oxidoreductase ERp57. Folding and disulfide bond formation within the heavy chain occurs prior to ␤ 2 m binding. There are four N-linked glycans on the CD1d heavy chain, and we mutated them individually to ascertain their importance for the assembly and function of CD1d-␤ 2 m heterodimers. None of the four were indispensable for assembly or the ability to bind ␣-galactosyl ceramide and to present it to human NKT cells. Nor were any required for the CD1d molecule to bind and present ␣-galactosyl ceramide after lysosomal processing of a precursor lipid, galactosyl-(␣1-2)-galactosyl ceramide. However, one glycan, glycan 2 at Asn-42, proved to be of particular importance for the stability of the CD1d-␤ 2 m heterodimer. A mutant CD1d heavy chain lacking glycan 2 assembled with ␤ 2 m and transported from the ER more rapidly than wild-type CD1d and dissociated more readily from ␤ 2 m upon exposure to detergents. A mutant expressing only glycan 1 dissociated completely from ␤ 2 m upon exposure to the detergent Triton X-100, whereas a mutant expressing only glycan 2 at Asn-42 was more stable. In addition, glycan 2 was not processed efficiently to the complex form in mature wild-type CD1d molecules. Modeling the glycans on the published structure indicated that glycan 2 interacts significantly with both the CD1d heavy chain and ␤ 2 m, which may explain these unusual properties.The human CD1 5 (cluster of differentiation 1) family consists of five transmembrane glycoproteins encoded by linked genes (1). They are divided into two groups based on amino acid sequence homology; group 1 includes CD1a, -b, and -c, and group 2 consists of CD1d, the only isoform present in mice and rats. The fifth member of the family, CD1e, has an amino acid sequence intermediate between the two groups. CD1 heavy chains are structurally similar to MHC class I molecules and possess a short C-terminal cytosolic tail, a hydrophobic transmembrane region, and an extracellular region that interacts non-covalently with ␤ 2 -microglobulin (␤ 2 m). The role of CD1 molecules is to bind lipid antigens and present them to T cells, and the ␣1 and ␣2 domains of the extracellular region fold in a similar manner to the analogous domains in MHC class I molecules to generate the lipid binding site.CD1 heavy chain folding and association with ␤ 2 m occurs in the endoplasmic reticulum (ER). After exiting the ER, the assembled CD1 molecules pass through the secretory pathway and reach the plasma membrane. From there, with the exception of CD1a, they enter the endocytic system by adaptor protein (AP)-dependent internalization using tyrosine-based endocytic motifs (YXX⌽, X ϭ any amino acid and ⌽ ϭ bulky hydrophobic amino acid). Similar to MHC class II molecul...

Invariant natural killer T (iNKT) cells recognize self lipid antigens presented by CD1d molecules. The nature of the self-antigens involved in the development and maturation of iNKT cells is poorly defined. Lysophospholipids are self-antigens presented by CD1d that are generated through the action of phospholipases A1 and A2. Lysosomal phospholipase A2 (LPLA2, group XV phospholipase A2) resides in the endocytic system, the main site where CD1d antigen acquisition occurs, suggesting that it could be particularly important in CD1d function. We find that Lpla2 −/− mice show a decrease in iNKT cell numbers that is neither the result of a general effect on the development of lymphocyte populations nor of effects on CD1d expression. However, endogenous lipid antigen presentation by CD1d is reduced in the absence of LPLA2. Our data suggest that LPLA2 plays a role in the generation of CD1d complexes with thymic lipids required for the normal selection and maturation of iNKT cells.antigen processing | lipid storage disease C D1d molecules are β 2 -microglobulin (β 2 m)-associated MHC class I homologs that bind lipid antigens and recycle between the plasma membrane and endocytic compartments. Endogenous and exogenous lipid binding can occur during transit through the secretory pathway and in the endocytic system, which is the main site of antigen loading (1). CD1d-lipid complexes are recognized by natural killer T (NKT) cells, a subset of T cells that shares characteristics of both the innate and adaptive arms of the immune system. NKT cells rapidly respond to stimulation by antigenpresenting cells (APCs) and release cytokines that can alter the strength and character of immune responses. They do so by transactivating, for example, NK cells, CD4+ and CD8 + T cells, and B cells, and by shifting cytokine responses to or from T helper (Th)1, Th2, and Th17 profiles. NKT cells have been shown to participate in inflammation, autoimmunity, cancer, and infectious diseases (2-4), and are characterized by the expression of both T-cell receptor (TCR) and NK cell markers. There are two main types: type I or invariant (iNKT), which express a semi-invariant TCR (Vα14-Jα18 in mice and Vα24-Jα18 in humans, paired with a restricted set of TCR-β chains, Vβ8.2, -7, or -2 in mice and Vβ11 in humans), and type II or noninvariant NKT cells that express more diverse TCRs (5-13).iNKT cells arise from double-positive (DP; CD4

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