Owen, Barbara

Abstract The influence of TAP-MHC class I interactions on peptide binding to the class I heavy chain is assessed during TAP-dependent assembly using Kb-specific Abs that recognize conformational changes induced by assembly with β2-microglobulin (β2m) and by peptide binding. A significant portion (45%) of Kb molecules in TAP+, RMA-derived microsomes are associated with the TAP complex as measured by coimmunoisolation of Kb using anti-TAP1 Abs, while only 20% of the Kb heavy chain molecules are isolated as Kbβ2m complexes with the α-Kb-specific Abs, Y-3 or K-10-56. The amount of Kb isolated with Y-3 and K-10-56 increases in proportion to transport and binding of peptide to the Kb molecules within the RMA microsomes. In contrast, less than 5% of the Kb within TAP2-RMA-S microsomes associated with the remaining TAP1 subunit. However, greater than 60% of Kb heavy chain is isolated as K-10-56- and Y-3-reactive Kbβ2m complexes. We propose that a TAP-MHC class I interaction serves to stabilize the MHC class I:β2m complex in an immature conformation (Y-3 and K-10-56 nonreactive) prior to high affinity peptide binding, preventing the export of class I molecules complexed with low affinity peptide ligands from the ER.

Mammalian cells have evolved sophisticated DNA repair systems to correct mispaired or damaged bases and extra helical loops. However, we find that two DNA repair systems, the mismatch recognition system (MMR) and the base excision repair, are turned into a causative factor rather than a safeguard of genomic integrity. We have followed CAG expansion in germ cells and in somatic tissues of transgenic mice harboring a fragment of the human Huntington's disease (HD) gene. CAG expansion of the hHD allele is abrogated in both somatic cells and in germ cells of transgenic mice that lack Msh2. A complex involving Msh2 is thought to cause expansions by binding to, but failing to correct at the site of DNA breaks. Base excision repair may cause the break that needs to be repaired. Surprisingly, expansion in somatic cells only occurs at midlife of the mouse. These data indicate that some process associated with aging causes expansion and DNA breaks only in midlife. We have purified the MSH2/MSH3 enzymes and examined their biochemical properties. We find that key steps of the MMR system are impaired and that other and repair pathways may be called into play to repair the site.

AbstractBackground: Patients with multiple myeloma often have Bence Jones proteins composed of free monoclonal light chains of the κ or λ type in their urine. Usually, these light chains exist as monomeric or dimeric forms, but rarely, larger molecules, such as tetramers, have been reported in the serum.Methods and Results: We report the presence of trimeric complexes of λ light chain dimers in a patient who was diagnosed with a free λ light chain multiple myeloma 2 years earlier and subsequently underwent a stem cell transplant. Recently, the patient presented with a large serum M-spike (23 g/L) by protein electrophoresis. The spike consisted of monoclonal λ light chains without a heavy chain. The urine contained only 8 mg of λ light chain in a 24-h specimen. Quantitative analysis of the serum and urinary free light chains (FLCs) indicated the probability of larger aggregates of FLCs. Size-exclusion chromatography, electrophoresis, analytical ultracentrifugation, and mass spectrometric studies of the serum revealed almost exclusively the presence of trimolecular aggregates of λ light chain dimers without other multimeric species.Conclusion: Monoclonal λ light chains may present as hexameric aggregates that cannot be cleared by renal excretion.

Association of heparin non-covalently with bovine factor Xa was analyzed by Superose-12 gel chromatography. In 0.05 M NaCl, 0.02 M Tris, pH 7.5, DEGR-Xa (factor Xa inactivated by dans-Glu-Gly-Arg-CH2Cl) was eluted as a single, sharp peak at Ve/Vt=0-65 (elution volume/internal volume). Mixtures of heparin and DEGR-Xa were eluted as two partially resolved peaks of protein at Ve/Vt=0.59 and 0.65. The fraction of DEGFUXa in the leading peak was directly proportional to [heparin], and at 100 yM heparin the leading peak contained more than half the total protein. When 0.02 M HEPES was substituted for Tris a single, slightly broadened peak at Ve/Vt=0.64 was obtained on chromatography of 100 μM heparin and 10 μM DEGR-Xa. In a buffer system comprising 0.02 M Tris, 0.02 M HEPES, 0.03 M NaCl, pH 7.5, two peaks were eluted at Ve/Vt=0.59 and 0.65. Therefore, Tris increases the affinity of DEGR-Xa for heparin.Solutions buffered with Tris or HEPES were compared for effects on the kinetics of inhibition of factor Xa by antithrombin III-heparin. Reaction mixtures containing 1 nM factor Xa, 30 nM heparin and 600 nM antithrombin III were assayed with S-2222 at intervals of 2-10 sec. Reagent concentrations were chosen (a) to assure pseudo-first-order kinetics, (b) to have [heparin]<< Kq for factor Xa-heparin, and (c) to bind virtually all available heparin to antithrombin III. The same second-order rate constant, Kobs=2.5×107 M−1s−1, was obtained in both buffer systems. We conclude that the association of factor Xa with heparin observed directly by gel chromatography does not contribute to the reaction rate of factor Xa with antithrombin III-heparin.

Abstract Once MHC class I heavy chain binds β2-microglobulin (β2m) within the endoplasmic reticulum, an assembly complex comprising the class I heterodimer, TAP, TAPasin, calreticulin, and possibly Erp57 is formed before the binding of high affinity peptide. TAP-dependent delivery of high affinity peptide to in vitro translated Kbβ2m complexes within microsomes (TAP+/TAPasin+) was studied to determine at which point peptide binding becomes resistant to thermal denaturation. It was determined that the thermal stability of Kb-β2m-peptide complexes depends on the timing of peptide binding to Kbβ2m relative to TAP binding high affinity peptide. Premature exposure of the TAP complex to high affinity peptide before its association with class I heavy chain results in Kbβ2m-peptide-TAP complexes that lose peptide upon exposure to elevated temperature after solubilization away from microsome-associated proteins. These findings suggest that the order in which class I heavy chain associates with endoplasmic reticulum-resident chaperones and peptide determines the stability of Kbβ2m-peptide complexes.