Andrea Setini

Distinctive Features of the α1-Domain a Helix of HLA-C Heavy Chains Free of β2-Microglobulin

Only a few monoclonal antibodies are available with a restricted specificity to HLA-C products. In the present report, we demonstrate that antibody L31, previously shown to react with beta 2m-less (free) class I MHC heavy chains, binds to an epitope (residues 66-68 of the alpha 1 domain alpha helix) present on all the HLA-C alleles corresponding to the accepted (CW1 through CW8) serologic specificities, and on a few HLA-B heavy chains sharing with HLA-C an aromatic residue at position 67. Extensive IEF blot testing of HLA homozygous, EBV-transformed B-lymphoid cells indicates that HLA-C molecules are present at significantly lower levels than HLA-B polypeptides not only at cell surface, as previously demonstrated, but also in total cellular extracts. Testing of metabolically labeled HLA-CW1, -CW5, and -CW6 transfectants and HLA homozygous lymphoid cells, particularly HLA-CW1-expressing cells, demonstrates that the L31 epitope is present on a subpopulation of naturally occurring HLA-C molecules distinct from that identified by antibody W6/32 to beta 2m-associated heavy chains. Pulse-chase experiments demonstrate that this epitope is transiently made available to antibody binding at early biosynthetic stages, but becomes hidden upon assembly with beta 2m. Thus, free HLA-C and other Y/F67+ heavy chains are characterized by distinctive antibody binding features in a region (residues 66-68) included in a previously identified HLA-C restricted motif, which has been suggested to be the primary cause of distinctive features of the antigen-binding groove, low affinity for endogenous peptide antigens and beta 2m, and preferential uptake of exogenous peptides, possibly of viral origin. We also show that HLA-CW1 heavy chains, both free and beta 2m associated, acquire sialilation. Free HLA-CW1 heavy chains are expressed at the cell surface even when unsialilated, albeit at low levels.

A Single Bottleneck in HLA-C Assembly

Poor assembly of class I major histocompatibility HLA-C heavy chains results in their intracellular accumulation in two forms: free of and associated with their light chain subunit (β2-microglobulin). Both intermediates are retained in the endoplasmic reticulum by promiscuous and HLA-dedicated chaperones and are poorly associated with peptide antigens. In this study, the eight serologically defined HLA-C alleles and the interlocus recombinant HLA-B46 allele (sharing the HLA-C-specific motif KYRV at residues 66–76 of the α1-domain α-helix) were compared with a large series of HLA-B and HLA-A alleles. Pulse-labeling experiments with HLA-C transfectants and HLA homozygous cell lines demonstrated that KYRV alleles accumulate as free heavy chains because of both poor assembly and post-assembly instability. Reactivity with antibodies to mapped linear epitopes, co-immunoprecipitation experiments, and molecular dynamics simulation studies additionally showed that the KYRV motif confers association to the HLA-dedicated chaperones TAP and tapasin as well as reduced plasticity and unfolding in the peptide-binding groove. Finally, in vitro assembly experiments in cell extracts of the T2 and 721.220 mutant cell lines demonstrated that HLA-Cw1 retains the ability to form a peptide-receptive interface despite a lack of TAP and functional tapasin, respectively. In the context of the available literature, these results indicate that a single locus-specific biosynthetic bottleneck renders HLA-C peptide-selective (rather than peptide-unreceptive) and a preferential natural killer cell ligand.

Conformation and surface expression of free HLA-CW1 heavy chains in the absence of β2-microglobulin

A beta 2-microglobulin (beta 2m)-deficient kidney carcinoma cell line and three monoclonal antibodies to the alpha 1 (L31), alpha 2 (W6/32), and alpha 3 (Q1/28) domain of class I HLA molecules were selected to assess the role of beta 2m in regulating the conformation and surface expression of HLA-C molecules. HLA-A2, -B27, and -CW1 molecules synthesized by beta 2m-deficient cells were compared to heavy chains synthesized in transfectants expressing a large excess of beta 2m. As assessed by differential binding with monoclonal antibodies and partitioning studies in the detergent TX-114, no HLA-A2, -B27, or -CW1 molecules can be expressed, in a correct conformation, by beta 2m-deficient cells. These cells, however, do express low but significant amounts of free HLA-CW1 heavy chains at the cell surface. Transfection with beta 2m causes a coordinate change in the antibody reactivity of the three domains of HLA-CW1 molecules, thereby providing the first experimental demonstration that assembly with beta 2m affects the folding of not only the alpha 1 and alpha 2, but also of the alpha 3 domain. HLA-CW1 heavy chains, when free of beta 2m, are less soluble in the detergent TX-114 than free HLA-B27 heavy chains, and when associated with beta 2m share an alpha 3 domain epitope with free HLA-A2 and -B27 heavy chains. Moreover, their assembly with beta 2m is largely incomplete. Those data additionally demonstrate an impaired ability of HLA-CW1 to properly fold and establish a close similarity of HLA-CW1 to murine Db and Ld molecules. Although the functional role, if any, of free HLA-CW1 heavy chains remains to be determined, the present study demonstrates that the absence of beta 2m does not completely ablate class I expression in neoplastic cells of human origin.

Impaired Assembly Results in the Accumulation of Multiple HLA-C Heavy Chain Folding Intermediates

Class I MHC H chains assemble with β2-microglobulin (β2m) and are loaded with peptide Ags through multiple folding steps. When free of β2m, human H chains react with Abs to linear epitopes, such as L31. Immunodepletion and coimmunoprecipitation experiments, performed in this study, detected a preferential association of L31-reactive, β2m-free H chains with calnexin in β2m-defective cells, and with calreticulin and TAP in β2m-expressing cells. In β2m-defective cells, the accumulation of calnexin-bound H chains stoichiometrically exceeded their overall accumulation, a finding that supports both chaperoning preferences and distinct sorting abilities for different class I folds. No peptide species, in a mass range compatible with that of the classical class I ligands, could be detected by mass spectrometry of acidic eluates from L31-reactive HLA-Cw1 H chains. In vitro assembly experiments in TAP-defective T2 cells, and in cells expressing an intact Ag-processing machinery, demonstrated that L31 H chains are not only free of, but also unreceptive to, peptides. L31 and HC10, which bind nearly adjacent linear epitopes of the α1 domain α helix, reciprocally immunodepleted free HLA-C H chains, indicating the existence of a local un-/mis-folding involving the N-terminal end of the α1 domain α helix and peptide-anchoring residues of the class I H chain. Thus, unlike certain murine free H chains, L31-reactive H chains are not the immediate precursors of conformed class I molecules. A model inferring their precursor-product relationships with other known class I intermediates is presented.

Effect of short-time exposures to nickel and lead on brain monoamine oxidase from Danio rerio and Poecilia reticulata

The aim of this work was to verify, in two small size freshwater teleosts Danio rerio and Poecilia reticulata, the effects of short‐time exposures (24 and 72 h) to a sublethal dose (500 μg/L) of nickel and lead, on brain monoamine oxidase (MAO), an important neural enzyme. The 24‐h treatment using both metals caused a strong reduction of MAO activity in D. rerio brain, whereas causing a slight MAO activity stimulation in P. reticulata brain. The same treatment in both species did not affect the brain MAO mRNA production as showed by RT‐PCR. Extending the duration of treatment as far as 72 h, partly (D. rerio) or completely (P. reticulata) reversed the metal effects on brain MAO activity suggesting that mechanisms to neutralize the metals had been activated.

N-Linked Glycosylation Selectively Regulates the Generic Folding of HLA-Cw1

To resolve primary (glycosylation-assisted) from secondary (glycosylation-independent) quality control steps in the biosynthesis of HLA (human leukocyte antigen) class I glycoproteins, the unique N-linked glycosylation site of the HLA-Cw1 heavy chain was deleted by site-directed mutagenesis. The non-glycosylated Cw1S88G mutant was characterized by flow cytometry, pulse-chase, co-immunoprecipitation, and in vitro assembly assays with synthetic peptide ligands upon transfection in 721.221 and 721.220 cells. The former provide a full set of primary as well as secondary chaperoning interactions, whereas the latter are unable to perform secondary quality control (e.g. proper class I assembly with peptide antigens) as a result of a functional defect of the HLA-dedicated chaperone tapasin. In both transfectants, Cw1S88G displayed a loss/weakening in its generic chaperoning interaction with calreticulin and/or ERp57 and became redistributed toward calnexin, known to bind the most unfolded class I conformers. Despite this, and quite unexpectedly, a weak interaction with the HLA-dedicated chaperone TAP was selectively retained in 721.221. In addition, the ordered, stepwise acquisition of thermal stability/peptide binding was disrupted, resulting in a heterogeneous ensemble of Cw1S88G conformers with unorthodox and unprecedented peptide assembly features. Because a lack of glycosylation and a lack of tapasin-assisted peptide loading have distinct, complementary, and additive effects, the former is separable from (and upstream of) the latter, e.g. primary quality control is suggested to supervise a crucial, generic folding step preliminary to the acquisition of peptide receptivity.

HLA-A, -B, -C genotyping and expression in human nonlymphoid tumor cell lines.

A combination of molecular genotyping and protein biochemistry methods was used to assess the HLA-A, -B, -C genotyping and expression of six tumor cell lines. Four cell lines had been previously HLA typed by conventional serologic methods. Two could not be typed by serology because deficient in the surface expression of HLA-A, -B, -C molecules. As shown herein, all the 25 alleles carried by the six tested cell lines were typed at the DNA level. In addition, discrepancies between the previous serologic and the present DNA typing results were detected in 9 of the 21 tested serologic specificities. Typing at the protein level by isoelectric focusing and allele-specific monoclonal antibodies confirmed the DNA typing data. Our results exemplify the limits of the serologic typing procedures and demonstrate that molecular methods are highly desirable to conduct functional experiments and identify HLA losses in neoplastic cells at single allele level.

Genotoxicity biomarkers in the amphipod Gammarus elvirae exposed in vivo to mercury and lead, and basal levels of DNA damage in two cell types

ABSTRACT The present work deals with the application of genotoxicity biomarkers by means of the Comet assay in haemocytes and spermatozoa of the crustacean Gammarus elvirae exposed in vivo to heavy metals. Furthermore, a basal levels (BLs) study of DNA damage in the two cell types considered for two different gammarids species, G. elvirae and Echinogammarus veneris, was carried out. It is important to identify factors that influence the outcome of the assay in order to obtain reliable and reproducible results usable for risk assessment purposes. Our results highlight that the Italian legal limits for Hg and Pb, respectively, 0.5 and 50 µg/L, are inadequate for establishing safety thresholds in the aquatic environment. Furthermore, the freshwater invertebrate G. elvirae, used for the first time to measure the effect of genotoxicants, is a good candidate for evaluating the genotoxicity damage induced by heavy metals. Our results concerning spermatozoa show excessively variable responses and high BLs.