Specificity of amyloid precursor-like protein 2 interactions with MHC class I molecules

The ubiquitously expressed amyloid precursor-like protein 2 (APLP2) has been previously found to regulate cell surface expression of the major histocompatibility complex (MHC) class I molecule K(d) and bind strongly to K(d). In the study reported here, we demonstrated that APLP2 binds, in varied degrees, to several other mouse MHC class I allotypes and that the ability of APLP2 to affect cell surface expression of an MHC class I molecule is not limited to K(d). L(d), like K(d), was found associated with APLP2 in the Golgi, but K(d) was also associated with APLP2 within intracellular vesicular structures. We also investigated the effect of beta(2)m on APLP2/MHC interaction and found that human beta(2)m transfection increased the association of APLP2 with mouse MHC class I molecules, likely by affecting H2 class I heavy chain conformation. APLP2 was demonstrated to bind specifically to the conformation of L(d) having folded outer domains, consistent with our previous results with K(d) and indicating APLP2 interacts with the alpha1alpha2 region on each of these H2 class I molecules. Furthermore, we observed that binding to APLP2 involved the MHC alpha3/transmembrane/cytoplasmic region, suggesting that conserved as well as polymorphic regions of the H2 class I molecule may participate in interaction with APLP2. In summary, we demonstrated that APLP2's binding, co-localization pattern, and functional impact vary among H2 class I molecules and that APLP2/MHC association is influenced by multiple domains of the MHC class I heavy chain and by beta(2)m's effects on the conformation of the heavy chain.     

The amyloid precursor-like protein 2 associates with the major histocompatibility complex class I molecule K(d)

Amyloid precursor-like protein 2 (APLP2) is a member of a protein family related to the amyloid precursor protein, which is implicated in Alzheimer's disease. Little is known about the physiological function of this protein family. The adenovirus E3/19K protein binds to major histocompatibility complex (MHC) class I antigens in the endoplasmic reticulum, thereby preventing their transport to the cell surface. In cells coexpressing E3/19K and the MHC K(d) molecule, K(d) is associated with E3/19K and two cellular protein species with masses of 100 and 110 kDa, termed p100/110. Interestingly, p100/110 are released from the complex upon the addition of K(d)-binding peptides, suggesting a role for these proteins in peptide transfer to MHC molecules. Here we demonstrate by microsequencing, reactivity with APLP2-specific antibodies, and comparison of biochemical parameters that p100/110 is identical to human APLP2. We further show that the APLP2/K(d) association does not require the physical presence of E3/19K. Thus, APLP2 exhibits an intrinsic affinity for the MHC K(d) molecule. Similar to the binding of MHC molecules to the transporter associated with antigen processing, complex formation between APLP2 and K(d) strictly depends upon the presence of beta(2)-microglobulin. Conditions that prolong the residency of K(d) in the endoplasmic reticulum lead to a profound increase of the association and a drastic reduction of APLP2 transport. Therefore, this unexpected interplay between these unrelated molecules may have implications for both MHC antigen and APLP2 function.     

Amyloid precursor-like protein 2 increases the endocytosis, instability, and turnover of the H2-K(d) MHC class I molecule

The defense against the invasion of viruses and tumors relies on the presentation of viral and tumor-derived peptides to CTL by cell surface MHC class I molecules. Previously, we showed that the ubiquitously expressed protein amyloid precursor-like protein 2 (APLP2) associates with the folded form of the MHC class I molecule K(d). In the current study, APLP2 was found to associate with folded K(d) molecules following their endocytosis and to increase the amount of endocytosed K(d). In addition, increased expression of APLP2 was shown to decrease K(d) surface expression and thermostability. Correspondingly, K(d) thermostability and surface expression were increased by down-regulation of APLP2 expression. Overall, these data suggest that APLP2 modulates the stability and endocytosis of K(d) molecules.     

Amyloid precursor-like protein 2 association with HLA class I molecules

Amyloid precursor-like protein 2 (APLP2) is a ubiquitously expressed protein. The previously demonstrated functions for APLP2 include binding to the mouse major histocompatibility complex (MHC) class I molecule H-2K^d and down regulating its cell surface expression. In this study, we have investigated the interaction of APLP2 with the human leukocyte antigen (HLA) class I molecule in human tumor cell lines. APLP2 was readily detected in pancreatic, breast, and prostate tumor lines, although it was found only in very low amounts in lymphoma cell lines. In a pancreatic tumor cell line, HLA class I was extensively co-localized with APLP2 in vesicular compartments following endocytosis of HLA class I molecules. In pancreatic, breast, and prostate tumor lines, APLP2 was bound to the HLA class I molecule. APLP2 was found to bind to HLA-A24, and more strongly to HLA-A2. Increased expression of APLP2 resulted in reduced surface expression of HLA-A2 and HLA-A24. Overall, these studies demonstrate that APLP2 binds to the HLA class I molecule, co-localizes with it in intracellular vesicles, and reduces the level of HLA class I molecule cell surface expression.     

Structural definition of the H-2Kd peptide-binding motif

Classic major histocompatibility complex (MHC) proteins associate with antigen- and self-derived peptides in an allele-specific manner. Herein we present the crystal structure of the MHC class I protein H-2K(d) (K(d)) expressed by BALB/c mice in complex with an antigenic peptide derived from influenza A/PR/8/34 nucleoprotein (Flu, residues 147-155, TYQRTRALV). Analysis of our structure in conjunction with the sequences of naturally processed epitopes provides a comprehensive understanding of the dominant K(d) peptide-binding motif. We find that Flu residues Tyr(P2), Thr(P5), and Val(P9) are sequestered into the B, C, and F pockets of the K(d) groove, respectively. The shape and chemistry of the polymorphic B pocket make it an optimal binding site for the side chain of Tyr(P2) as the dominant anchoring residue of nonameric peptides. The non-polar F pocket limits the amino acid repertoire at P9 to hydrophobic residues such as Ile, Leu, or Val, whereas the C pocket restricts the size of the P5-anchoring side chain. We also show that Flu is accommodated in the complex through an unfavorable kink in the otherwise extended peptide backbone due to the presence of a prominent ridge in the K(d) groove. Surprisingly, this backbone conformation is strikingly similar to D(b)-presented peptides despite the fact that these proteins employ distinct motif-anchoring strategies. The results presented in this study provide a solid foundation for the understanding of K(d)-restricted antigen presentation and recognition events.     

Amyloid precursor-like protein 2 promotes cell migration toward fibronectin and collagen IV.

Previous studies have established that in response to wounding, the expression of amyloid precursor-like protein 2 (APLP2) in the basal cells of migrating corneal epithelium is greatly up-regulated. To further our understanding of the functional significance of APLP2 in wound healing, we have measured the migratory response of transfected Chinese hamster ovary (CHO) cells expressing APLP2 isoforms to a variety of extracellular matrix components including laminin, collagen types I, IV, and VII, fibronectin, and heparan sulfate proteoglycans (HSPGs). CHO cells overexpressing either of two APLP2 variants, differing in chondroitin sulfate (CS) attachment, exhibit a marked increase in chemotaxis toward type IV collagen and fibronectin but not to laminin, collagen types I and VII, and HSPGs. Cells overexpressing APLP2-751 (CS-modified) exhibited a greater migratory response to fibronectin and type IV collagen than their non-CS-attached counterparts (APLP2-763), suggesting that CS modification enhanced APLP2 effects on cell migration. Moreover, in the presence of chondroitin sulfate, transfectants overexpressing APLP2-751 failed to exhibit this enhanced migration toward fibronectin. The APLP2-ECM interactions were also explored by solid phase adhesion assays. While overexpression of APLP2 isoforms moderately enhanced CHO adhesion to laminin, collagen types I and VII, and HSPGs lines, especially those overexpressing APLP2-751, exhibited greatly increased adhesion to type IV collagen and fibronectin. These observations suggest that APLP2 contributes to re-epithelialization during wound healing by supporting epithelial cell adhesion to fibronectin and collagen IV, thus influencing their capacity to migrate over the wound bed. Furthermore, APLP2 interactions with fibronectin and collagen IV appear to be potentiated by the addition of a CS chain to the core proteins.     

The endoplasmic reticulum lumenal domain of the adenovirus type 2 E3-19K protein binds to peptide-filled and peptide-deficient HLA-A*1101 molecules

E3-19K is a type I membrane glycoprotein expressed by adenoviruses (Ads) to modulate host antiviral immune responses. We have developed an expression system for the endoplasmic reticulum lumenal domain (residues 1 to 100) of Ad type 2 E3-19K tagged with a C-terminal His6 sequence in baculovirus-infected insect cells. In this system, recombinant E3-19K is secreted into the culture medium. A characterization of soluble E3-19K by analytical ultracentrifugation and circular dichroism showed that the protein is monomeric and adopts a stable and correctly folded tertiary structure. Using a gel mobility shift assay and analytical ultracentrifugation, we showed that soluble E3-19K associates with soluble peptide-filled and peptide-deficient HLA-A*1101 molecules. This is the first example of a viral immunomodulatory protein that interacts with conformationally distinct forms of class I major histocompatibility complex molecules. The E3-19K/HLA-A*1101 complexes formed in a 1:1 stoichiometry with equilibrium dissociation constants (Kd) of 50 +/- 10 nM for peptide-filled molecules and of about 10 microM for peptide-deficient molecules. A temperature-dependent proteolysis study revealed that the association of E3-19K with peptide-deficient HLA-A*1101 molecules stabilizes the binding groove. Importantly, our studies showed that peptide-deficient HLA-A*1101 molecules sequestered by E3-19K are capable of binding antigenic peptides and maturing into peptide-filled molecules. This firmly establishes that E3-19K does not block binding of antigenic peptides. Together, our results suggest that Ads have evolved to exploit the late and early stages of the class I antigen presentation pathway.     

Allele- and locus-specific recognition of class I MHC molecules by the immunomodulatory E3-19K protein from adenovirus

The E3-19K protein from human adenoviruses (Ads) retains class I MHC molecules in the endoplasmic reticulum. As a consequence, the cell surface expression of class I molecules is suppressed, allowing Ads to evade immune surveillance. Using native gel electrophoresis, gel filtration chromatography, and surface plasmon resonance, we show that a soluble form of the Ad type 2 (Ad2) E3-19K protein associates with HLA-A and -B molecules; equilibrium dissociation constants were in the nanomolar range and approximately 2.5-fold higher affinity for HLA-A (-A*0201, -A*0301, -A*1101, -A*3301, and -Aw*6801) relative to HLA-B (-B*0702 and -B*0801) molecules. Among the alleles of the HLA-A locus examined, HLA-A*3101 associated approximately 15-fold less avidly with soluble E3-19K. Soluble E3-19K interacted only very weakly with HLA-Cw*0304, and no interaction with HLA-Cw*0401 could be detected under identical conditions. Site-directed mutagenesis and flow cytometry demonstrated that MHC residue 56 plays a critical role in the association and endoplasmic reticulum retention of HLA-A molecules by E3-19K. This delineates the spatial environment around residue 56 as a putative E3-19K interaction surface on class I molecules. Overall, our data imply that a link may exist between host genetic factors and the susceptibility of individuals to Ad infections.     

The E3/19K protein of adenovirus type 2 binds to the domains of histocompatibility antigens required for CTL recognition

The E3/19K protein of human adenovirus type 2 binds to HLA class I antigens and blocks their terminal glycosylation and cell surface expression. The nature of this interaction is non-covalent and involves neither disulfide bridges between the two molecules nor their carbohydrates. The murine H-2 Kd antigen associates with the E3/19K protein in a similar fashion to human HLA antigens whereas the allelic product H-2 Kk does not. Hybrid genes between the Kd and Kk alleles were constructed and their products were expressed in embryonic kidney cells together with the E3/19K protein. This allowed us to identify the alpha 1 and alpha 2 domains as the essential structures of the histocompatibility antigens for binding the viral protein. Interestingly, these domains are also crucial for T cell recognition. The implications for the evolution of adenoviruses and their ability to cause persistent infections are discussed.     

Adenovirus E3-19K proteins of different serotypes and subgroups have similar, yet distinct, immunomodulatory functions toward major histocompatibility class I molecules

Our understanding of the mechanism by which the E3-19K protein from adenovirus (Ad) targets major histocompatibility complex (MHC) class I molecules for retention in the endoplasmic reticulum is derived largely from studies of Ad serotype 2 (subgroup C). It is not well understood to what extent observations on the Ad2 E3-19K/MHC I association can be generalized to E3-19K proteins of other serotypes and subgroups. The low levels of amino acid sequence homology between E3-19K proteins suggest that these proteins are likely to manifest distinct MHC I binding properties. This information is important as the E3-19K/MHC I interaction is thought to play a critical role in enabling Ads to cause persistent infections. Here, we characterized interaction between E3-19K proteins of serotypes 7 and 35 (subgroup B), 5 (subgroup C), 37 (subgroup D), and 4 (subgroup E) and a panel of HLA-A, -B, and -C molecules using native gel, surface plasmon resonance (SPR), and flow cytometry. Results show that all E3-19K proteins exhibited allele specificity toward HLA-A and -B molecules; this was less evident for Ad37 E3-19K. The allele specificity for HLA-A molecules was remarkably similar for different serotypes of subgroup B as well as subgroup C. Interestingly, all E3-19K proteins characterized also exhibited MHC I locus specificity. Importantly, we show that Lys(91) in the conserved region of Ad2 E3-19K targets the C terminus of the α2-helix (MHC residue 177) on MHC class I molecules. From our data, we propose a model of interaction between E3-19K and MHC class I molecules.     

Requirements for the association of adenovirus type 2 E3/19K wild-type and mutant proteins with HLA antigens

The E3/19K protein of human adenovirus type 2 is a resident of the endoplasmic reticulum (ER). Immediately after synthesis, it associates with major histocompatibility complex class I antigens and prevents their intracellular transport and cell surface expression. We have generated several C-terminal deletion mutants of the E3/19K protein that are preterminated at various positions on both sides of the membrane-spanning segment of the protein. One of these mutants is terminated at the luminal side of the membrane (M310), and two are terminated in the hydrophobic segment (M374 and M392), whereas mutant M621 is terminated on the cytoplasmic side of the ER membrane. The M310, M374, and M392 mutants are soluble proteins. They do not associate with HLA antigens in transfected 293 cells, and they are, to some extent, secreted into the medium. The M621 mutant protein is integrated in the ER membrane, associates immediately after its synthesis with HLA antigens, and exits from the ER. By using either an in vitro translation system supplemented with microsomes or overexpression in insect cells, we showed that M374 and E3/19K are able to associate with HLA antigens. These results indicate that the conformation of the luminal part of the E3/19K protein is not grossly altered by the mutations. Rapid transport of the M374 mutant out of the ER and partial degradation of this protein may prevent the interaction with HLA class I antigens in transfected 293 cells.     

Abrogation of cell surface expression of human class I transplantation antigens by an adenovirus protein in Xenopus laevis oocytes

Class I transplantation antigens form complexes with a virus protein encoded in the early region E3 of the adenovirus-2 genome. The interaction between this viral glycoprotein, E19, and nascent human class I antigens has been examined by microinjecting purified mRNA into Xenopus laevis oocytes. Both E19 and the two class I antigen subunits, the heavy chain and beta 2-microglobulin (beta 2M), were efficiently translated. The heavy chains did not become terminally glycosylated, as monitored by endoglycosidase H digestion, and were not expressed on the oocyte surface unless they were associated with beta 2M. The E19 protein did not become terminally glycosylated, and we failed to detect this viral protein on the surface of the oocytes. Co-translation of heavy chain and E19 mRNA demonstrated that the two proteins associate intracellularly. However, neither protein appeared to be transported to the trans-Golgi compartment. Similar observations were made in adenovirus-infected HeLa cells. Heavy chains bound to beta 2M became terminally glycosylated in oocytes in the presence of low concentrations of E19. At high concentrations of the viral protein, no carbohydrate modifications and no cell surface expression of class I antigens were apparent. Thus, beta 2M and E19 have opposite effects on the intracellular transport of the heavy chains. These data suggest that adenovirus-2 may impede the cell surface expression of class I antigens to escape immune surveillance.     

An adenovirus type 2 glycoprotein blocks cell surface expression of human histocompatibility class I antigens

The adenovirus type 2 encoded protein E3/19K binds to human histocompatibility class I antigens (HLA). This association occurs both in adenovirus-infected cells and in cells that have been transfected with the gene encoding the E3/19K protein. The formation of the HLA-E3/19K complex prevents the HLA antigens from being correctly processed by inhibiting their terminal glycosylation. This effect is specific for HLA antigens and does not generally involve the glycosyltransferases. Furthermore, the HLA-E3/19K association dramatically reduces the cell surface expression of the HLA antigens. This reduced level of antigens might influence the cytotoxic T cell response. Therefore, our results show a possible molecular mechanism whereby adenoviruses, and perhaps other viruses, delay or escape the cellular immune system of the host.     

Kb, Kd, and Ld molecules share common tapasin dependencies as determined using a novel epitope tag

The endoplasmic reticulum protein tapasin is considered to be a class I-dedicated chaperone because it facilitates peptide loading by proposed mechanisms such as peptide editing, endoplasmic reticulum retention of nonpeptide-bound molecules, and/or localizing class I near the peptide source. Nonetheless, the primary functions of tapasin remain controversial as do the relative dependencies of different class I molecules on tapasin for optimal peptide loading and surface expression. Tapasin dependencies have been addressed in previous studies by transfecting different class I alleles into tapasin-deficient LCL721.220 cells and then monitoring surface expression and Ag presentation to T cells. Indeed, by these criteria, class I alleles have disparate tapasin-dependencies. In this study, we report a novel and more direct method of comparing tapasin dependency by monitoring the ratio of folded vs open forms of the different mouse class I heavy chains, L(d), K(d), and K(b). Furthermore, we determine the amount of de novo heavy chain synthesis required to attain comparable expression in the presence vs absence of tapasin. Our findings show that tapasin dramatically improves peptide loading of all three of these mouse molecules.     

APLP1 and APLP2, members of the APP family of proteins,behave similarly to APP in that they associate with NMDA receptors and enhance NMDA receptor surface expression

The function of amyloid precursor protein (APP) is unknown, although the discovery that it contributes to the regulation of surface expression of N‐methyl‐d ‐aspartate (NMDA) receptors has afforded new insights into its functional significance. Since APP is a member of a gene family that contains two other members, amyloid precursor‐like proteins 1 and 2 (APLP1 and APLP2), it is important to determine if the related APP proteins possess the same properties as APP with respect to their interactions with NMDA receptors. Following expression in mammalian cells, both APLP1 and APLP2 behaved similarly to APP in that they both co‐immunoprecipitated with the two major NMDA receptor subtypes, GluN1/GluN2A and GluN1/GluN2B, via interaction with the obligatory GluN1 subunit. Immunoprecipitations from detergent extracts of adult mammalian brain showed co‐immunoprecipitation of APLP1 and APLP2 with GluN2A‐ and GluN2B‐containing NMDA receptors. Furthermore, similarly to APP, APLP1 and APLP2 both enhanced GluN1/GluN2A and GluN1/GluN2B cell surface expression. Thus, all the three members of the APP gene family behave similarly in that they each contribute to the regulation of cell surface NMDA receptor homoeostasis.

     

Mechanism for Amyloid Precursor-like Protein 2 Enhancement of Major Histocompatibility Complex Class I Molecule Degradation

Earlier studies have demonstrated interaction of the murine major histocompatibility complex (MHC) class I molecule K^d with amyloid precursor-like protein 2 (APLP2), a ubiquitously expressed member of the amyloid precursor protein family. Our current findings indicate that APLP2 is internalized in a clathrin-dependent manner, as shown by utilization of inhibitors of the clathrin pathway. Furthermore, we demonstrated that APLP2 and K^d bind at the cell surface and are internalized together. The APLP2 cytoplasmic tail contains two overlapping consensus motifs for binding to the adaptor protein-2 complex, and mutation of a tyrosine shared by both motifs severely impaired APLP2 internalization and ability to promote K^d endocytosis. Upon increased expression of wild type APLP2, K^d molecules were predominantly directed to the lysosomes rather than recycled to the plasma membrane. These findings suggest a model in which APLP2 binds K^d at the plasma membrane, facilitates uptake of K^d in a clathrin-dependent manner, and routes the endocytosed K^d to the lysosomal degradation pathway. Thus, APLP2 has a multistep trafficking function that influences the expression of major histocompatibility complex class I molecules at the plasma membrane.     

Novel Zinc-binding Site in the E2 Domain Regulates Amyloid Precursor-like Protein 1 (APLP1) Oligomerization

The amyloid precursor protein (APP) and the APP-like proteins 1 and 2 (APLP1 and APLP2) are a family of multidomain transmembrane proteins possessing homo- and heterotypic contact sites in their ectodomains. We previously reported that divalent metal ions dictate the conformation of the extracellular APP E2 domain (Dahms, S. O., Könnig, I., Roeser, D., Gührs, K.-H., Mayer, M. C., Kaden, D., Multhaup, G., and Than, M. E. (2012) J. Mol. Biol. 416, 438–452), but unresolved is the nature and functional importance of metal ion binding to APLP1 and APLP2. We found here that zinc ions bound to APP and APLP1 E2 domains and mediated their oligomerization, whereas the APLP2 E2 domain interacted more weakly with zinc possessing a less surface-exposed zinc-binding site, and stayed monomeric. Copper ions bound to E2 domains of all three proteins. Fluorescence resonance energy transfer (FRET) analyses examined the effect of metal ion binding to APP and APLPs in the cellular context in real time. Zinc ions specifically induced APP and APLP1 oligomerization and forced APLP1 into multimeric clusters at the plasma membrane consistent with zinc concentrations in the blood and brain. The observed effects were mediated by a novel zinc-binding site within the APLP1 E2 domain as APLP1 deletion mutants revealed. Based upon its cellular localization and its dominant response to zinc ions, APLP1 is mainly affected by extracellular zinc among the APP family proteins. We conclude that zinc binding and APP/APLP oligomerization are intimately linked, and we propose that this represents a novel mechanism for regulating APP/APLP protein function at the molecular level.     

Specificity of the mouse cytotoxic T lymphocyte response to adenovirus 5. E1A is immunodominant in H-2b, but not in H-2d or H-2k mice

The Ag specificity and MHC restriction of the CTL response to adenovirus 5 (Ad5) in three strains of mice, C57BL/10 (H-2b), BALB/c (H-2d), and C3H/HeJ (H-2k), were tested. Polyclonal Ad5-specific CTL were prepared by priming mice in vivo with live Ad5 virus followed by secondary in vitro stimulation of the spleen cells with virus-infected syngeneic cells. The Ad5-specific CTL were Db restricted in C57BL/10 and Kk restricted in C3H/HeJ. In BALB/c mice both Kd- and Dd/Ld-restricted CTL were detected. The polyclonal Ad5-specific CTL response in C57BL/10 mice is directed exclusively against the products of the E1A region, which comprises only 5% of the Ad5 genome. In BALB/c mice E1A is at best a very minor target Ag and in C3H/HeJ mice E1A is not recognized at all. Using the H-2 congenic mouse strains B10.BR (H-2k) and C3H.SW (H-2b) it was shown that the immunodominance of E1A is H-2 dependent. The 19-kDa glycoprotein encoded in the E3 region of Ad5, which binds to class I MHC in the endoplasmic reticulum and prevents its translocation to the cell surface, does not affect the specificity of the CTL response in C57BL/10 mice toward E1A. However, it affects the MHC restriction of the Ad5-specific response in BALB/c mice, selectively inhibiting generation of Kd-restricted CTL.