Peptide binding to HLA-DR1: a peptide with most residues substituted to alanine retains MHC binding

Major histocompatibility complex (MHC) glycoproteins play an important role in the development of an effective immune response. An important MHC function is the ability to bind and present 'processed antigens' (peptides) to T cells. We show here that the purified human class II MHC molecule, HLA-DR1, binds peptides that have been shown to be immunogenic in vivo. Detergent-solubilized HLA-DR1 and a papain-cleaved form of the protein lacking the transmembrane and intracellular regions have similar peptide binding properties. A total of 39 single substitutions were made throughout an HLA-DR1 restricted hemagglutinin epitope and the results determine one amino acid in this peptide which is crucial to binding. Based on this analysis, a synthetic peptide was designed containing two residues from the original hemagglutinin epitope embedded in a chain of polyalanine. This peptide binds to HLA-DR1, indicating that the majority of peptide side chains are not required for high affinity peptide binding.     

Immobilized human hemoglobin,a versatile matrix for analytical and biotechnological applications

The analytical and biotechnological applications of human hemoglobin immobilized covalently on CNBr–Sepharose 4B are reviewed. Hemoglobin is bound to the matrix as αβ dimers via either chain. The immobilized αβ dimers maintain the capacity to interact reversibly with soluble ones under conditions where the soluble protein is in self-association equilibrium. Under these conditions, therefore, immobilized dimers bind part of the soluble protein. In turn, the binding process can be used to assess the specific features of the equilibrium on solid-phase and to extract selectively hemoglobin from a variety of biological specimens of practical interest. A different application of immobilized αβ dimers concerns their use in the determination of the equilibrium and kinetic stability of the heme–globin linkage, a property that is directly correlated with the stability of the hemoglobin molecule. The advantages and limitations attendant the use of the immobilized protein relative to the soluble one are discussed.     

Assembly of HLA DR1 molecules translated in vitro: binding of peptide in the endoplasmic reticulum precludes association with invariant chain.

MHC class II molecules usually bind peptides in the endocytic pathway, but can also present endogenous peptides from newly synthesized proteins in a chloroquine-insensitive manner, suggesting that peptide binding might occur in the endoplasmic reticulum (ER). We used in vitro translation of HLA-DR1 class II molecules in the presence of microsomes to study peptide binding in the ER. Formation of functional class II molecules in vitro depends on formation of disulfide bridges in alpha and beta chains. The class II alpha beta heterodimers made by in vitro translation resemble class II molecules synthesized in cells in (i) their reactivity with conformation-specific antibodies, (ii) their assembly with Ii chain homotrimers, (iii) the generation of SDS-stable dimers upon peptide binding and (iv) their specificity of peptide binding. The assembly of class II molecules occurs via an alpha beta intermediate and can occur post-translationally, but only in intact microsomes. Class II alpha beta heterodimers are able to bind peptides in ER-derived microsomes, a process that precludes subsequent association of class II molecules with Ii chain. This mechanism might explain presentation of endogenous peptides by class II molecules.     

Transport and intracellular distribution of MHC class II molecules and associated invariant chain in normal and antigen-processing mutant cell lines

We have compared the intracellular transport and subcellular distribution of MHC class II-invariant chain complexes in a wild-type HLA-DR3 homozygous cell line and a mutant cell line, T2.DR3. The latter has a defect in antigen processing and accumulates HLA-DR3 molecules associated with an invariant chain-derived peptide (CLIP) rather than the normal complement of peptides derived from endocytosed proteins. We find that in the wild-type cells, CLIP is transiently associated with HLA-DR3 molecules, suggesting that the peptide is a normal class II- associated intermediate generated during proteolysis of the invariant chain. In the mutant cell line proteolysis of the invariant chain is less efficient, and HLA-DR3/CLIP complexes are generated much more slowly. Examination of the mutant cell line by immunoelectronmicroscopy shows that class II-invariant chain complexes accumulate intracellularly in large acidic vesicles which contain lysosomal markers, including beta-hexosaminidase, cathepsin D, and the lysosomal membrane protein CD63. The markers in these vesicles are identical to those seen in the class II-containing vesicles (MIICs) seen in the wild- type cells but the morphology is drastically different. The vesicles in the mutant cells are endocytic, as measured by the internalization of BSA-gold conjugates. The implication of these findings for antigen processing in general and the nature of the mutation in particular are discussed.     

A role for calnexin (IP90) in the assembly of class II MHC molecules.

Major histocompatibility complex (MHC) class II antigens consist of alpha and beta chains that associate intracellularly with the invariant (I) chain. The HLA-DR alpha beta I complex assembles in the endoplasmic reticulum (ER) into a nonameric structure via progressive addition of three alpha beta dimers to a core invariant chain trimer. We have examined intracellular association of alpha beta I complexes with the resident ER protein calnexin. Calnexin associates rapidly (within 3 min) with newly synthesized alpha, beta and I chains, and remains associated with the assembling alpha beta I complex until the final alpha beta dimer is added, forming the complete nonamer. Dissociation of calnexin parallels egress of alpha beta I from the ER. These results suggest that calnexin retains and stabilizes both free class II subunits and partially assembled class II-I chain complexes until assembly of the nonamer is complete.     

Stable surface expression of invariant chain prevents peptide presentation by HLA-DR.

Class II major histocompatibility complex (MHC) molecules are cell surface glycoproteins that bind and present immunogenic peptides to T cells. Intracellularly, class II molecules associate with a polypeptide referred to as the invariant (Ii) chain. Ii is proteolytically degraded and dissociates from the class II complex prior to cell surface expression of the mature class II alpha beta heterodimer. Using human fibroblasts transfected with HLA-DR1 and Ii cDNAs, we now demonstrate that truncation of the cytoplasmic domain of Ii results in the failure of Ii to dissociate from the alpha beta Ii complex and leads to stable expression of class II alpha beta Ii complexes on the cell surface. Furthermore, biochemical analysis and peptide presentation assays demonstrated that transfectants with stable surface alpha beta Ii complexes expressed very few free alpha beta heterodimers at the surface and were very inefficient in their ability to present immunogenic peptides to T cells. These results support the hypothesis that the cytoplasmic domain of Ii is responsible for endosomal targeting of alpha beta Ii and directly demonstrate that association with Ii interferes with the antigen presentation function of class II molecules.     

Bone marrow pre-B lymphocytes synthesize immunoglobulin mu chains of membrane type with different properties and intracellular pathways.

Mouse normal bone marrow pre-B lymphocytes synthesize only membrane mu chains (micron), as shown by mRNA studies and peptide analysis. The micron chains exist in two forms: free micron chains assembled into dimers, or L chain-bound micron chains present in IgM monomers (in the case of 'late pre-B cells', i.e., after productive L chain gene rearrangement). These two forms of molecules are very different in properties, fate and intracellular pathways. Free but not L chain-bound mu chains are highly susceptible to mild proteolysis, which degrades their entire Cmu 1 and VH domains. Free mu chains are rapidly degraded within the lysosomal compartment, which they reach via the cis, avoiding the trans, part of the Golgi complex. In contrast, as soon as mu chains bind to L chains, they are directed towards the 'trans' Golgi compartment, where they undergo terminal glycosylation, then to the cell surface, where they progressively accumulate. It is suggested that the conformation instability of the Cmu 1 and VH domains of the free mu chains plays a critical role in the intracellular targeting of these molecules, as compared with that of L chain-bound mu chains.     

HLA-DP, HLA-DQ, and HLA-DR have different requirements for invariant chain and HLA-DM

The MHC is central to the adaptive immune response. The human MHC class II is encoded by three different isotypes, HLA-DR, -DQ, and -DP, each being highly polymorphic. In contrast to HLA-DR, the intracellular assembly and trafficking of HLA-DP molecules have not been studied extensively. However, different HLA-DP variants can be either protective or risk factors for infectious diseases (e.g. hepatitis B), immune dysfunction (e.g. berylliosis), and autoimmunity (e.g. myasthenia gravis). Here, we establish a system to analyze the chaperone requirements for HLA-DP and to compare the assembly and trafficking of HLA-DP, -DQ, and -DR directly. Unlike HLA-DR1, HLA-DQ5 and HLA-DP4 can form SDS-stable dimers supported by invariant chain (Ii) in the absence of HLA-DM. Uniquely, HLA-DP also forms dimers in the presence of HLA-DM alone. In model antigen-presenting cells, SDS-stable HLA-DP complexes are resistant to treatments that prevent formation of SDS-stable HLA-DR complexes. The unexpected properties of HLA-DP molecules may help explain why they bind to a more restricted range of peptides than other human MHC class II proteins and frequently present viral peptides.     

Structural and functional properties of αβ-heterodimers of tropomyosin with myopathic mutations Q147P and K49del in the β-chain

Tropomyosin (Tpm) is an α-helical coiled-coil actin-binding protein that plays a key role in the Ca²⁺-regulated contraction of striated muscles. Two Tpm isoforms, α (Tpm 1.1) and β (Tpm 2.2), are expressed in fast skeletal muscles. These Tpm isoforms can form either αα and ββ homodimers, or αβ heterodimers. However, only αα-Tpm and αβ-Tpm dimers are usually present in most of fast skeletal muscles, because ββ-homodimers are relatively unstable and cannot exist under physiologic conditions. Nevertheless, the most of previous studies of myopathy-causing mutations in the Tpm β-chains were performed on the ββ-homodimers. In the present work, we applied different methods to investigate the effects of two myopathic mutations in the β-chain, Q147P and K49del (i.e. deletion of Lys49), on structural and functional properties of Tpm αβ-heterodimers and to compare them with the properties of ββ-homodimers carrying these mutations in both β-chains. The results show that the properties of αβ-Tpm heterodimers with these mutations in the β-chain differ significantly from the properties of ββ-homodimers with the same substitutions in both β-chains. This indicates that the αβ-heterodimer is a more appropriate model for studying the effects of myopathic mutations in the β-chain of Tpm than the ββ-homodimer which virtually does not exist in human skeletal muscles.     

MHC class II binding of peptides derived from HLA-DR 1.

The aim of these studies was to determine whether auto- and alloreactivity can arise from T cell recognition of MHC-peptides in context of syngeneic MHC. Four synthetic peptides derived from the first domain of the HLA-DR beta 1 * 0101 chain were used in limiting dilution analysis to prime T cells from HLA-DR1- and HLA-DR1+ responders. The frequency of T cells responding to these four peptides was similar in individuals with or without HLA-DR1. In both cases, the peptide corresponding to the nonpolymorphic sequence 43-62, was less immunogenic than peptides corresponding to the three hypervariable regions 1-20, 21-42, and 66-90, eliciting a lower number of reactive T cells. Experiments using a T cell line with specific reactivity to peptide 21-42 showed, however, that this response can be efficiently blocked by adding to the culture a nonpolymorphic sequence peptide. This suggests that alloreactivity can be blocked by use of monomorphic (self) peptides. The binding of both "monomorphic" and "polymorphic" synthetic DR1 peptides to affinity purified HLA-DR 1 and DR 11 molecules was measured using radiolabeled peptides and high performance size exclusion chromatography. The data showed that the polymorphic as well as monomorphic synthetic DR1 peptides bound to both DR1 and DR11 molecules. Competitive inhibition studies indicated that the monomorphic 43-62 peptide can block the binding of the polymorphic peptides, consistent with the results obtained in T cell cultures. Taken together these data suggest that anti-MHC autoreactive T cells are present in the periphery and that both auto and alloreactivity can be elicited by MHC peptides binding to MHC class II molecules.     

Conformational and structural characteristics of peptides binding to HLA-DR molecules.

A fundamental characteristic of MHC class I and class II proteins is their unusual capacity to form stable complexes with a wide spectrum of peptide ligands. In this study, sets of peptide analogues containing long chain-biotinylated lysine individually substituted for each amino acid in the sequence have been used to explore the structural requirements for the formation of peptide-MHC class II protein complexes. Based on the ability of the analogs to bind both the MHC protein and fluorescent streptavidin, receptor contact residues were identified and from their spacing the conformation of the bound peptides could be inferred. Six separate peptides were studied; three defined by HLA-DR1Dw1-restricted T cells, and three identified by T cells restricted through alleles other than HLA-DR1Dw1. The similar patterns of fluorescent signals observed when the former three peptides were studied indicated that they shared conformational features when bound to HLA-DR1Dw1. In contrast when the latter three peptides were examined, the data indicated that they shared some but not all of the conformational features characteristic of the peptides known to elicit HLA-DR1Dw1-restricted T cells. When the peptide sequences were aligned based on the critical contact residues, two positions of structural homology were apparent. In each sequence, an amino acid with a bulky hydrophobic side chain could be identified separated by four residues from a small amino acid. These minimal structural requirements were consistent with recent experiments demonstrating that only a small number of side chains in the peptide were necessary for binding to the MHC protein.     

Invariant chain protects class II histocompatibility antigens from binding intact polypeptides in the endoplasmic reticulum.

Unlike class I histocompatibility (MHC) antigens, most newly synthesized MHC class II molecules fail to be loaded with peptides in the endoplasmic reticulum (ER), binding instead to the invariant chain glycoprotein (Ii). Ii blocks the class II peptide binding groove until the class II:Ii complexes are transported to endosomes where Ii is removed by proteolysis, thus permitting loading with endosomal short peptides (approximately 12-25 amino acids). Ligands from which the groove is protected by Ii have not yet been identified; theoretically they could be short peptides or longer polypeptides (or both), because the class II groove is open at both ends. Here we show that in Ii- deficient cells, but not in cells expressing large amounts of Ii, a substantial fraction of class II alpha beta dimers forms specific, SDS-resistant 1:1 complexes with a variety of polypeptides. Different sets of polypeptides bound to H-2Ak, Ek, Ed and HLA-DR1 class II molecules; for Ak, a major species of Mr 50 kDa (p50) and further distinct 20 and 130 kDa polypeptides were detectable. Class II binding of p50 was characterized in detail. Point mutations within the Ak antigen binding groove destabilized the p50:class II complexes; a mutation outside the groove had no effect. A short segment of p50 was sufficient for association with Ak. The p50 polypeptide was synthesized endogenously, bound to Ak in a pre-Golgi compartment, and was transported to the cell surface in association with Ak. Thus, Ii protects the class II groove from binding endogenous, possibly misfolded polypeptides in the ER. The possibility is discussed that polypeptide binding is an ancestral function of the MHC antigen binding domain.     

Primary structure of the reactive site of human C1-inhibitor

Human C1-inhibitor (C1-Inh) forms an equimolar complex with complement proteinase C1s that is resistant to dissociation by sodium dodecyl sulfate. The formation of this stable complex results in the cleavage of a peptide bond near the carboxyl terminus of the inhibitor and, whereas the bulk of C1-Inh remains covalently bound to the light chain of C1s, the postcomplex inhibitor peptide can be isolated under denaturing conditions. We have sequenced the amino-terminal region of this peptide and deduced that it represents the carboxyl-terminal side of the reactive site of C1-Inh. Limited proteolysis of C1-Inh by Crotalus atrox protease results in an active derivative lacking an amino-terminal peptide of 36 residues. Further proteolysis of this derivative with Pseudomonas aeruginosa elastase inactivates the inhibitor and a peptide is released. The amino-terminal sequence of this peptide overlaps with that of the postcomplex peptide and indicates that the residue imparting primary specificity to the inhibitor is arginine.     

Point mutations in or near the antigen-binding groove of HLA-DR3 implicate class II-associated invariant chain peptide affinity as a constraint on MHC class II polymorphism

During maturation of MHC II molecules, newly synthesized and assembled complexes of MHC II alphabeta dimers with invariant chain (Ii) are targeted to endosomes, where Ii is proteolyzed, leaving remnant class II-associated Ii peptides (CLIP) in the MHC II peptide binding groove. CLIP must be released, usually with assistance from the endosomal MHC II peptide exchange factor, HLA-DM, before MHC II molecules can bind endosomal peptides. Structural factors that control rates of CLIP release remain poorly understood, although peptide side chain-MHC II specificity pocket interactions and MHC II polymorphism are important. Here we report that mutations betaS11F, betaS13Y, betaQ70R, betaK71E, betaK71N, and betaR74Q, which map to the P4 and P6 pockets of the groove of HLA-DR3 molecules, as well as alphaG20E adjacent to the groove, are associated with elevated CLIP in cells. Most of these mutations increase the resistance of CLIP-DR3 complexes to dissociation by SDS. In vitro, the groove mutations increase the stability of CLIP-DR3 complexes to dissociation. Dissociation rates in the presence of DM, as well as coimmunoprecipitation of some mutant DR3 molecules with DM, are also diminished. The profound phenotypes associated with some of these point mutations suggest that the need to maintain efficient CLIP release represents a constraint on naturally occurring MHC II polymorphism.     

Delineating the interaction of combretastatin A-4 with αβ tubulin isotypes present in drug resistant human lung carcinoma using a molecular modeling approach

Abstract

Tubulin isotypes are known to regulate microtubule dynamic instability and contribute to the development of drug resistance in certain types of cancers. Combretastatin-A4 (CA-4) has a potent anti-mitotic, vascular disrupting and anti-angiogenic activity. It binds at the interface of αβ tubulin heterodimers and inhibits microtubules assembly. Interestingly, the CA-4 resistant human lung carcinoma shows alteration of βI and βIII isotype levels, a higher expression of βI tubulin isotype and a decreased expression of βIII tubulin isotypes has been reported in drug resistant cell lines. However, the origin of CA-4 resistance in lung carcinoma is not well understood. Here, we investigate the interaction and binding affinities of αβI, αβIIb, αβIII and αβIVa tubulin isotypes with CA-4, employing molecular modeling approaches. Sequence analysis shows that variations in residue composition at the CA-4 binding pocket of βI, βIII and βIVa tubulin isotypes when compared to template βIIb isotype. Molecular docking result shows that the CA-4 prefers ‘cis’ conformation in all αβ-tubulin isotypes. Molecular dynamics simulation reveal role of H7 helix, T7 loop and H8 helix of β-tubulin in lower binding affinity of αβI and αβIII isotypes for CA-4. The order of binding energy for CA-4 is αβIIb?>?αβIVa?>?αβI?>?αβIII. This suggest that drug resistance is induced in human lung carcinoma cells by altering the expression of β-tubulin isotypes namely βI and βIII which show lowest binding affinities. Our present study can help in designing potential CA-4 analogs against drug-resistant cancer cells showing altered expression of tubulin isotypes. Abbreviations: CA-4 combretastatin-A4

MD molecular dynamics

RMSD root mean square deviation

DSSP dictionary of secondary structure of proteins

VMD visual molecular dynamics

Communicated by Ramaswamy H. Sarma     

Protein-protein interactions:possible location of hemoglobin-haptoglobin contacts

We have studied the binding of α and β chain tryptic peptides to Hp linked to agarose. The results obtained provide evidence for a specific binding of peptides to Hp. Addition of Hb, which binds irreversibly Hp resulted in a specific displacement of the peptides bound to Hp. The analysis of the peptides displaced and their location in the Hb molecule suggested that Hb interacts with Hp through surfaces by which (I) αβ dimers and (II) α and β monomers face each other in the Hb tetramer.     

Rapid nonlysosomal degradation of assembled HLA class II glycoproteins incorporating a mutant DR alpha-chain

Gamma irradiation followed by antibody and complement selection was used to isolate a human B-lymphoblastoid cell line that no longer expresses HLA-DR molecules on its cell surface. Cell surface expression in the mutant (HMy2.DRN) was restored by transfecting a wildtype DRA but not a DRB cDNA, suggesting that a structural mutation in the DRA mRNA or protein was responsible for the lack of cell surface expression. Nucleotide sequence analysis of the DRA mRNA from HMy2.DRN revealed a 75 nucleotide deletion corresponding to the start of the alpha 2 domain and involving one of two cysteines that are involved in the formation of an intrachain disulfide bond. At the biochemical level, only minute quantities of HLA-DR could be precipitated from this cell line after a 4-h continuous label with 35S-methionine. HLA-DR beta and the class II-associated invariant chain could be seen coprecipitating with the mutant DR alpha-chain, suggesting a limited accumulation of normally assembled molecules. However, by carrying out the labeling at 16 degrees C instead of 37 degrees C, equivalent amounts of HLA-DR could be precipitated from parent and mutant alike. The mutant DR alpha chain was found in association with the beta-chain, but with reduced association with the invariant chain under these conditions. Pulse chase analysis in the parent and mutant cell lines indicated that this mutant DR alpha beta I complex undergoes a process of degradation at 37 degrees C. Inhibitors of intracellular transport such as monensin were ineffective in blocking this process of degradation. This work is consistent with published reports implicating the involvement of a pre-Golgi or an early Golgi compartment in the proteolysis of aberrantly folded or assembled multisubunit proteins.     

The attack of the phytopathogens and the trumpet solo: Identification of a novel plant antifungal peptide with distinct fold and disulfide bond pattern

Phytopathogens cause economic losses in agribusiness. Plant-derived compounds have been proposed to overcome this problem, including the antimicrobial peptides (AMPs). This paper reports the identification of Ps-AFP1, a novel AMP isolated from the Pisum sativum radicle. Ps-AFP1 was purified and evaluated against phytopathogenic fungi, showing clear effectiveness. In silico analyses were performed, suggesting an unusual fold and disulfide bond pattern. A novel fold and a novel AMP class were here proposed, the αβ-trumpet fold and αβ-trumpet peptides, respectively. The name αβ-trumpet was created due to the peptide's fold, which resembles the musical instrument. The Ps-AFP1 mechanism of action was also proposed. Microscopic analyses revealed that Ps-AFP1 could affect the fungus during the hyphal elongation from spore germination. Furthermore, confocal microscopy performed with Ps-AFP1 labeled with FITC shows that the peptide was localized at high concentration along the fungal cell surface. Due to low cellular disruption rates, it seems that the main target is the fungal cell wall. The binding thermogram and isothermal titration, molecular dynamics and docking analyses were also performed, showing that Ps-AFP1 could bind to chitin producing a stable complex. Data here reported provided novel structural–functional insights into the αβ-trumpet peptide fold.     

Kinetic mechanisms of Ca++/calmodulin dependent protein kinases

Many of the cellular responses to Ca⁺⁺ signaling are modulated by a family of multifunctional Ca⁺⁺/calmodulin dependent protein kinases (CaMKs): CaMK I, CaMK II and CaMK IV. In order to further understand the role of CaMKs, we investigated the kinetic mechanism of CaMK II isozymes in comparison with those of CaMK I and CaMK IV by analyzing their steady state kinetics using phospholamban as a phosphoacceptor. The results indicated that (a) the CaMK family’s reaction mechanisms were of the sequential type in which all substrates must bind to enzyme before any product is released; (b) CaMK I and CaMK IV exhibited random sequential mechanism where either phospholamban or ATP can bind to the free enzyme; (c) the data of product inhibition for CaMK IIs best fit with an Ordered Bi Bi mechanism in which phospholamban is the first substrate to bind and ADP is the last product to be released; and (d) the constant α (ratio of apparent dissociation constants for binding peptide in the presence and absence of the second ligand) of all isozymes for ATP and peptide was higher than 1 indicating that the binding of phospholamban to CaMK decreased the enzyme’s affinity toward ATP.