Biomedical subjects

E D Mellins

Publications and source records attributed to E D Mellins.

16 recordsLinked to original sources

PubMed2001

Rheumatoid arthritis (RA)-associated HLA-DR alleles form less stable complexes with class II-associated invariant chain peptide than non-RA-associated HLA-DR alleles.

Certain HLA-DR alleles confer strong susceptibility to the autoimmune disease rheumatoid arthritis (RA). We compared RA-associated alleles, HLA-DR*0401, HLA-DR*0404, and HLA-DR*0405, with closely related, non-RA-associated alleles, HLA-DR*0402 and HLA-DR*0403, to determine whether they differ in their interactions with the class II chaperone, invariant chain (Ii). Ii binds to class II molecules in the endoplasmic reticulum, inhibits binding of other ligands, and directs class II-Ii complexes to endosomes, where Ii is degraded to class II-associated Ii peptide (CLIP). To evaluate the interaction of Ii and CLIP with these DR4 alleles, we introduced HLA-DR*0401, *0402, and *0404 alleles into a human B cell line that lacked endogenous HLA-DR or HLA-DM molecules. In a similar experiment, we introduced HLA-DR*0403 and *0405 into an HLA-DM-expressing B cell line, 8.1.6, and its DM-negative derivative, 9.5.3. Surface abundance of DR4-CLIP peptide complexes and their susceptibility to SDS-induced denaturation suggested that the different DR4-CLIP complexes had different stabilities. Pulse-chase experiments showed CLIP dissociated more rapidly from RA-associated DR molecules in B cell lines. In vitro assays using soluble rDR4 molecules showed that DR-CLIP complexes of DR*0401 and DR*0404 were less stable than complexes of DR*0402. Using CLIP peptide variants, we mapped the reduced CLIP interaction of RA-associated alleles to the shared epitope region. The reduced interaction of RA-associated HLA-DR4 molecules with CLIP may contribute to the pathophysiology of autoimmunity in RA.

Alleles↗

PubMed2001

The kinetic basis of peptide exchange catalysis by HLA-DM.

The mechanism by which the peptide exchange factor HLA-DM catalyzes peptide loading onto structurally homologous class II MHC proteins is an outstanding problem in antigen presentation. The peptide-loading reaction of class II MHC proteins is complex and includes conformational changes in both empty and peptide-bound forms in addition to a bimolecular binding step. By using a fluorescence energy transfer assay to follow the kinetics of peptide binding to the human class II MHC protein HLA-DR1, we find that HLA-DM catalyzes peptide exchange by facilitating a conformational change in the peptide-bound complex, and not by promoting the bimolecular MHC-peptide reaction or the conversion between peptide-receptive and -averse forms of the empty protein. Thus, HLA-DM serves essentially as a protein-folding or conformational catalyst.

Antigens, Differentiation, B-Lymphocyte↗

PubMed2001

Autoantigenic HCgp39 epitopes are presented by the HLA-DM-dependent presentation pathway in human B cells.

It is hypothesized that autoimmune diseases manifest when tolerance to self-Ags fails. One possible mechanism to break tolerance is presentation of self-Ag in an altered form. Most Ags are presented by APCs via the traditional presentation pathway that includes "epitope editing" by intracellular HLA-DM, a molecule that selects for stable MHC-peptide complexes. We were interested in testing the hypothesis that autoreactive MHC-peptide complexes may reach the cell surface by an alternate pathway without being edited by HLA-DM. We selected a cartilage autoantigen human cartilage glycoprotein 39 to which T cell responses are observed in rheumatoid arthritis (RA) patients and some DR(*)04 healthy subjects. RA is genetically associated with certain DRB1 alleles, including DRB1(*)0401 but closely related allele DRB1(*)0402 is either neutral or mildly protective with respect to RA. We generated human B lymphoblastoid cell line cells expressing DR(*)0401 or DR(*)0402 in the presence or absence of intracellular HLA-DM and assessed their ability to present a candidate autoantigen, human cartilage glycoprotein 39. Our results show that the presence of intracellular HLA-DM is critical for presentation of this autoantigen to CD4(+) T cell hybridomas generated from DR(*)04-transgenic mice. Presentation of an autoantigen by the traditional HLA-DM-dependent pathway has implications for Ag presentation events in RA.

Adipokines↗

PubMed2000

pH stability of HLA-DR4 complexes with antigenic peptides.

Complexes between antigenic peptides and class II proteins of the major histocompatibility complex (MHC) trigger cellular immune responses. These complexes usually dissociate more rapidly at mildly acidic pH, where they are formed intracellularly, as compared to neutral pH, where they function at the cell surface. This paper describes the pH dependence of the dissociation kinetics of complexes between MHC proteins and antigenic peptides containing aspartic and glutamic acid residues. Some of these complexes show an unusual pH dependence, dissociating much more rapidly at pH 7 than at pH 5.3. This occurs when the carboxylate group of the aspartic or glutamic acid residue is located in a neutral pocket of the protein. In contrast, solvent-exposed carboxylate groups or carboxylate groups buried in pockets where they form salt bridges with the protein do not show this unusual pH dependence. The kinetic data having the unusual pH dependence conform closely to a model in which there is a rapid reversible equilibration between a less stable deprotonated complex and a more stable protonated complex. In this model, the pK(a) of the protonation reaction for the partially buried peptide carboxylate group ranges from 7.7 to 8.3, reflecting the strongly basic conditions required for deprotonation. One of the few peptide/MHC complexes demonstrated to play a role in autoimmunity in humans contains a buried peptide carboxylate and shows this unusual pH dependence. The relevance of this finding to understanding the chemical basis of autoimmunity is briefly discussed.

Adipokines↗

PubMed2000

Accessory molecules for MHC class II peptide loading.

Accessory molecules, such as HLA-DM and invariant chain, modulate the ligands bound to MHC class II molecules in antigen-presenting cells. Recent investigations, including gene targeting experiments, have shed light on the functions of these molecules, their mechanisms of action, interactions with class II molecules, and the relationships with associated molecules such as tetraspanins and HLA-DO.

Animals↗

PubMed2000

Determination of the HLA-DM interaction site on HLA-DR molecules.

HLA-DM removes CLIP and other loosely bound peptides from MHC class II molecules. The crystal structures of class II molecules and of HLA-DM have not permitted identification of their interaction sites. Here, we describe mutations in class II that impair interactions with DM. Libraries of randomly mutagenized DR3 alpha and beta chains were screened for their ability to cause cell surface accumulation of CLIP/DR3 complexes in EBV-B cells. Seven mutations were associated with impaired peptide loading in vivo, as detected by SDS stability assays. In vitro, these mutant DR3 molecules were resistant to DM-catalyzed CLIP release and showed reduced binding to DM. All mutations localize to a single lateral face of HLA-DR, which we propose interacts with DM during peptide exchange.

Amino Acid Sequence↗

PubMed1998

Secondary structure composition and pH-dependent conformational changes of soluble recombinant HLA-DM.

HLA-DM catalyzes the release of invariant chain fragments from newly synthesized major histocompatibility complex (MHC) class II molecules, stabilizes empty class II molecules, and edits class II-associated peptides by preferentially releasing those that are loosely bound. The ability of HLA-DM to carry out these functions in vitro is pH dependent, with an optimum at pH 4.5-5.5 and poor activity at pH 7. The structural basis for these properties of HLA-DM is unknown. Sequence homology suggests that HLA-DM resembles classical, peptide-binding MHC class II molecules. In this study, we examined whether HLA-DM has a secondary structure composition consistent with an MHC fold and whether HLA-DM changes conformation between pH 5 and pH 7. Far-UV circular dichroism (CD) spectra of recombinant soluble HLA-DM (sDM) indicate that HLA-DM belongs to the alpha/beta class of proteins and structurally resembles both MHC class I and class II molecules. The CD peak around 198 nm increases upon going from neutral to endosomal pH and drops sharply upon denaturation below pH 3.5, distinguishing at least three states of sDM: the denatured state and two highly similar folded states. Fluorescence emission spectra show a slight blue-shift and a approximately 20% drop in intensity at pH 5 compared with pH 7. Unfolding experiments using guanidinium chloride show that the stability of sDM is somewhat reduced but not lost at pH 5. These results indicate that sDM undergoes a pH-dependent conformational change between neutral and endosomal pH. The change seems to involve both hydrogen bonding patterns and the hydrophobic core of sDM and may contribute to the pH dependence of DM activity.

Circular Dichroism↗

PubMed1998

DR/CLIP (class II-associated invariant chain peptides) and DR/peptide complexes colocalize in prelysosomes in human B lymphoblastoid cells.

In APCs, MHC class II molecules (MHC class II) bind antigenic peptides after HLA-DM mediated removal of CLIP. To characterize intracellular sites of peptide loading in human B lymphoblastoid cell lines, we conducted immunoelectron microscopy studies with Abs recognizing MHC class II associated with CLIP or bound peptide, respectively, together with Abs to HLA-DM and endocytic markers. The distribution of these molecules indicates that peptide binding occurs in compartments with characteristics of normal late endosomes, and in compartments that show characteristics of late endosomes, but are not detectably accessed by endocytosed BSA-gold. The latter compartments may represent or give rise to recycling vesicles that deliver peptide-loaded class II molecules to the cell surface. In addition, we have compared cells in which HLA-DM and HLA-DR interaction is defective with cells in which this interaction is intact, and find that DM/DR interaction is not required for the proper localization of either molecule to peptide-loading compartments.

Antigens, Differentiation, B-Lymphocyte↗

PubMed1998

Novel glycosylation of HLA-DRalpha disrupts antigen presentation without altering endosomal localization.

The HLA-DR hemizygous B lymphoblastoid cell line, 10.24.6, has a DRA mutation (Pro96-->Ser) that creates a novel glycosylation site at Asn94. The mutant DR molecules are primarily associated with nested fragments of invariant chain (class II-associated invariant chain peptides), and their interaction with HLA-DM is impaired. Here we further analyzed the defect in 10.24.6 cells. Expressing Ser96 mutant DRA cDNA in DRA-null cells recapitulated the 10.24.6 phenotype, indicating that the mutation causes the Ag presentation defect. A mutation to Ala96alpha, which does not introduce an extra glycan, generated a normal phenotype; the critical role of the glycan was further supported by experiments in which N-glycosylation was blocked by tunicamycin. We also evaluated whether the 10.24.6 mutation affected DR3 maturation or trafficking. Metabolic labeling and subcellular fractionation showed that assembly, endosomal transport, and invariant chain proteolysis of mutant DR3 molecules were similar to wild-type. A slight delay in export from the endoplasmic reticulum to the Golgi apparatus in 10.24.6 cells probably did not contribute significantly to the Ag presentation defect, because the abundance of DM and mutant DR in peptide-loading compartments was normal at steady state. Our results indicate that proper localization of these molecules does not depend on their interaction.

Antigen Presentation↗

PubMed1998

Aberrant intermolecular disulfide bonding in a mutant HLA-DM molecule: implications for assembly, maturation, and function.

HLA-DM (abbreviated DM) is an MHC-encoded glycoprotein that catalyzes the selective release of peptides, including class II-associated invariant chain peptides, from MHC class II molecules. To perform its function, DM must assemble in the endoplasmic reticulum (ER), travel to endosomes, and interact productively with class II molecules. We have described previously an EBV-transformed B cell line, 7.12.6, which displays a partial Ag presentation defect and expresses a mutated DM beta-chain with Cys79 replaced by Tyr. In this study, we show that HLA-DR molecules in 7.12.6 have a defect in peptide loading and accumulate class II-associated invariant chain peptides (CLIP). Peptide loading is restored by transfection of wild-type DMB. The mutant DM molecules exit the ER slowly and are degraded rapidly, resulting in greatly reduced levels of mutant DM in post-Golgi compartments. Whereas wild-type DM forms noncovalent alphabeta dimers, such dimers form inefficiently in 7.12.6; many mutant DM beta-chains instead form a disulfide-bonded dimer with DM alpha. Homodimers of DM beta are also detected in 7.12.6 and in the alpha-chain defective mutant, 2.2.93. We conclude that during folding of wild-type DM, the native conformation is stabilized by a conserved disulfide bond involving Cys79beta and by noncovalent contacts with DM alpha. Without these interactions, DM beta can form malfolded structures containing interchain disulfide bonds; malfolding is correlated with ER retention and accelerated degradation.

Antigens, Differentiation, B-Lymphocyte↗

PubMed1996

Developing and shedding inhibitions: how MHC class II molecules reach maturity.

Over the past year, several important advances have been made in understanding the mechanisms by which class II MHC glycoproteins acquire endosomal peptides inside antigen-presenting cells. Recent progress in the study of class II antigen presentation includes the identification of ligands from which invariant chain protects class II molecules in pre-endosomal compartments, an improved understanding of how invariant chain inhibits antigenic peptide binding, and the appreciation that HLA-DM (a factor important for antigen presentation in vivo) can act as a catalyst for peptide exchange.

Antigen Presentation↗

PubMed1993

Predicting remission in juvenile rheumatoid arthritis with methotrexate treatment.

Forty-nine children with a polyarticular course of juvenile rheumatoid arthritis treated with methotrexate (MTX) for at least one year were analyzed to identify clinical characteristics that would predict remission of arthritis after MTX treatment. Twenty-two children (45%) had remission of arthritis after a mean of 13.6 months of treatment and did not differ from the 27 with persistently active arthritis regarding years of disease before starting MTX, age starting MTX, maximum MTX dose, disease onset type, presence of radiographic joint destruction, concomitant treatment with hydroxychloroquine, sulfasalazine or prednisone, or presence of rheumatoid factor or antinuclear antibodies. Higher dose MTX, earlier treatment, genetic markers, and a standardized route of therapy may yield important information in future studies.

Arthritis, Juvenile↗

PubMed1993

Eosinophilic fasciitis in children frequently progresses to scleroderma-like cutaneous fibrosis.

We evaluated longterm outcome in 21 pediatric patients with biopsy proven eosinophilic fasciitis (EF), 4 followed in our rheumatology clinic and 17 reported in the literature. Two-thirds of these patients developed residual cutaneous fibrosis, while one-third enjoyed complete resolution of disease. Children under age 7 years were twice as likely as those over age 7 years to experience disease progression to cutaneous fibrosis [relative risk = 2.0 (95% confidence intervals 1.2, 3.4)]. Fourteen of 17 patients with extensive disease at diagnosis (involvement of 3-4 extremities +/- trunk) progressed to cutaneous fibrosis whereas all 4 patients with minimal disease (involvement of 1-2 extremities) at onset resolved completely. We detected no association between progression to cutaneous fibrosis and sex of patient, duration of symptoms prior to therapy, type of therapy, history of prior physical stress, or laboratory variables at diagnosis.

Adolescent↗

PubMed1992

The role of the MHC in autoimmunity: an overview.

The basis for MHC associations with autoimmune diseases is still unknown; however, recent progress in the study of the MHC, its gene products and their functions suggests a number of possible mechanisms. These paradigms are described and their applicability to particular autoimmune diseases is considered.

Antigen-Presenting Cells↗

PubMed1991

Decreasing severity of chronic uveitis in children with pauciarticular arthritis.

We compared the current prevalence and severity of chronic uveitis in children with pauciarticular juvenile rheumatoid arthritis in Seattle, Wash, with that of children with the same condition in the same area in 1975. The prevalence of eye disease decreased from 45% in 1975 to 13% in 1989, and the proportion of patients with severe visual loss decreased from 21% in 1975 to none in 1989. We could not attribute these findings to differences in known risk factors for iritis, such as age, sex, or presence of antinuclear antibodies. There was no difference in the duration of follow-up between the two groups. It is possible that the decline in prevalence of uveitis reflects a referral bias for eye disease in the 1975 population. However, the decrease in disease severity remains unexplained and may represent more effective treatment, earlier surveillance for ocular disease, or a change in the frequency of ocular manifestations of this disease in the 1989 group.

Arthritis, Juvenile↗

PubMed1989

Lymphocyte activation provokes modification of a lymphocyte-specific protein tyrosine kinase (p56lck).

The protein tyrosine kinase p56lck is implicated in the control of lymphocyte growth by virtue of its overexpression in some lymphoid malignancies and its transforming activity in heterologous systems. Previous studies have demonstrated that levels of lck mRNA and of p56lck decline rapidly after T cell activation. The disappearance of p56lck results primarily from post-translational conversion of p56lck to more slowly migrating forms with apparent sizes of approximately 60 kDa. This modification can be provoked by treatment of lymphocytes with PMA, and has been associated with increased serine phosphorylation of the p56lck molecule. Here we demonstrate that conversion of p56lck to p60lck is a feature of the physiologic activation of T lymphocytes by antigen-presenting cells. In addition, we show that the PMA-induced modification of p56lck proceeds via a mechanism distinct from conventional protein kinase C activation. The rapid conversion of p56lck to p60lck after antigenic stimulation is consistent with the view that this membrane-associated protein tyrosine kinase regulates some aspects of the lymphocyte activation sequence.

Adult↗