Banff Histopathological Consensus Criteria for Preimplantation Kidney Biopsies.

The Banff working group on preimplantation biopsy was established to develop consensus criteria (best practice guidelines) for the interpretation of preimplantation kidney biopsies. Digitally scanned slides were used (i) to evaluate interobserver variability of histopathologic findings, comparing frozen sections with formalin-fixed, paraffin-embedded tissue of wedge and needle core biopsies, and (ii) to correlate consensus histopathologic findings with graft outcome in a cohort of biopsies from international medical centers. Intraclass correlations (ICCs) and univariable and multivariable statistical analyses were performed. Good to fair reproducibility was observed in semiquantitative scores for percentage of glomerulosclerosis, arterial intimal fibrosis and interstitial fibrosis on frozen wedge biopsies. Evaluation of frozen wedge and core biopsies was comparable for number of glomeruli, but needle biopsies showed worse ICCs for glomerulosclerosis, interstitial fibrosis and tubular atrophy. A consensus evaluation form is provided to help standardize the reporting of histopathologic lesions in donor biopsies. It should be recognized that histologic parameters may not correlate with graft outcome in studies based on organs deemed to be acceptable after careful clinical assessment. Significant limitations remain in the assessment of implantation biopsies.

Immune responses to collagen-IV and fibronectin in renal transplant recipients with transplant glomerulopathy.

Antibodies (Abs) to donor HLA (donor-specific antibodies [DSA]) have been associated with transplant glomerulopathy (TG) following kidney transplantation (KTx). Immune responses to tissue-restricted self-antigens (self-Ags) have been proposed to play a role in chronic rejection. We determined whether KTx with TG have immune responses to self-Ags, Collagen-IV (Col-IV) and fibronectin (FN). DSA were determined by solid phase assay, Abs against Col-IV and FN by enzyme-linked immunosorbent assay and CD4+ T cells secreting interferon gamma (IFN-γ), IL-17 or IL-10 by ELISPOT. Development of Abs to self-Ags following KTx increased the risk for TG with an odds ratio of 22 (p-value = 0.001). Abs to self-Ags were IgG and IgM isotypes. Pretransplant Abs to self-Ags increased the risk of TG (22% vs. 10%, p < 0.05). Abs to self-Ags were identified frequently in KTx with DSA. TG patients demonstrated increased Col-IV and FN specific CD4+ T cells secreting IFN-γ and IL-17 with reduction in IL-10. We conclude that development of Abs to self-Ags is a risk factor and having both DSA and Abs to self-Ags increases the risk for TG. The increased frequency of self-Ag-specific IFN-γ and IL-17 cells with reduction in IL-10 demonstrate tolerance breakdown to self-Ags which we propose play a role in the pathogenesis of TG.

Differentiation of human bone marrow mesenchymal stem cells on decellularized extracellular matrix materials.

Mesenchymal bone marrow stromal cells may be a source of cells to preseed decellularized biologic mesh materials for improved cellularization and promote a more physiologic tissue after remodeling. Spontaneous differentiation of mesenchymal stromal cells on the decellularized material would be undesirable. Conversely, induced differentiation of mesenchymal stem cells (MSC) on the material would suggest that these materials may have promise as scaffold materials for bone, cartilage, or adipocyte formation. Two sources of mesenchymal cells were evaluated for induced differentiation in control wells. These MSCs were also evaluated for spontaneous or induced differentiation on decellularized porcine dermis and mesothelium materials. Primarily harvested bone marrow MSCs and commercially obtained MSCs were induced into osteoblasts and adipocytes on decellularized dermis and mesothelium materials. The MSCs were able to be induced into chondrocytes in pellet form but not when grown as a monolayer on the materials. The MSCs did not undergo spontaneous differentiation when grown on the materials for up to four weeks. MSC grown on decellularized porcine dermis or mesothelium do not spontaneously differentiate and may serve as a source of autologous cells for preseeding these extracellular matrix materials prior to implantation.

X11alpha modulates secretory and endocytic trafficking and metabolism of amyloid precursor protein: mutational analysis of the YENPTY sequence.

The neuronal adaptor X11alpha interacts with the conserved -GYENPTY- sequence in the C-terminus of amyloid precursor protein (APP) or its Swedish mutation (APPswe) to inhibit Abeta40 and Abeta42 secretion. We hypothesized that the -YENP- motif essential for APP endocytosis is also essential for X11alpha-mediated effects on APP trafficking and metabolism, and that X11alpha modulates APP metabolism in both secretory and endocytic pathways. X11alpha failed to interact with the endocytic-defective APPswe mutants Y738A, N740A, or P741A, and thus did not modulate their trafficking or metabolism. However, endocytic-competent APPswe Y743A had unique trafficking and metabolism including a prolonged half-life and increased secretion of catabolites compared with APPswe. In contrast to endocytic-defective mutants, X11alpha interacted with APPswe Y743A as well as with APPswe. Thus, similar to APPswe, coexpression of X11alpha with APPswe Y743A retarded its maturation, prolonged its half-life, and inhibited APPs, Abeta40, and Abeta42 secretion. Collectively, these data suggest that by direct interaction with the APPswe -YENP- motif in the cytoplasmic tail, X11alpha modulated its trafficking and processing in both secretory and endocytic compartments, and may reduce secretion of Abeta generated in either pathway.

Mechanisms for oxidizing low-density lipoprotein. Insights from patterns of oxidation products in the artery wall and from mouse models of atherosclerosis.

The oxidation hypothesis proposes that oxidative modification of low density lipoprotein (LDL) plays a critical role in atherogenesis. This review critically evaluates the various mechanisms proposed for LDL oxidation, focusing on insights derived from chemical analysis of human artery wall and studies of genetically engineered mice. The implications of recent clinical trials of vitamin E for the oxidation hypothesis are also briefly discussed.

Neutrophils employ the myeloperoxidase system to generate antimicrobial brominating and chlorinating oxidants during sepsis.

The myeloperoxidase system of neutrophils uses hydrogen peroxide and chloride to generate hypochlorous acid, a potent bactericidal oxidant in vitro. In a mouse model of polymicrobial sepsis, we observed that mice deficient in myeloperoxidase were more likely than wild-type mice to die from infection. Mass spectrometric analysis of peritoneal inflammatory fluid from septic wild-type mice detected elevated concentrations of 3-chlorotyrosine, a characteristic end product of the myeloperoxidase system. Levels of 3-chlorotyrosine did not rise in the septic myeloperoxidase-deficient mice. Thus, myeloperoxidase seems to protect against sepsis in vivo by producing halogenating species. Surprisingly, levels of 3-bromotyrosine also were elevated in peritoneal fluid from septic wild-type mice and were markedly reduced in peritoneal fluid from septic myeloperoxidase-deficient mice. Furthermore, physiologic concentrations of bromide modulated the bactericidal effects of myeloperoxidase in vitro. It seems, therefore, that myeloperoxidase can use bromide as well as chloride to produce oxidants in vivo, even though the extracellular concentration of bromide is at least 1,000-fold lower than that of chloride. Thus, myeloperoxidase plays an important role in host defense against bacterial pathogens, and bromide might be a previously unsuspected component of this system.

The protease inhibitor, MG132, blocks maturation of the amyloid precursor protein Swedish mutant preventing cleavage by beta-Secretase.

Amyloid (Abeta) peptides found aggregated into plaques in Alzheimer's disease are derived from the sequential cleavage of the amyloid precursor protein (APP) first by beta- and then by gamma-secretases. Peptide aldehydes, which inhibit cysteine proteases and proteasomes, reportedly block Abeta peptide secretion by interfering with gamma-secretase cleavage. Using a novel, specific, and sensitive enzyme-linked immunosorbent assay for the beta-secretase-cleaved fragment of the Swedish mutant of APP (APPSw), we determined that the peptide aldehyde, MG132, prevented beta-secretase cleavage. This block in beta-secretase cleavage was not observed with clasto-lactacystin beta-lactone and thus, cannot be attributed to proteasomal inhibition. MG132 inhibition of beta-secretase cleavage was compared with the serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF). AEBSF inhibition of beta-secretase cleavage was immediate and did not affect alpha-secretase cleavage. With MG132, inhibition was delayed and it decreased secretion of alpha-cleaved APPSw as well. Furthermore, MG132 treatment impaired maturation of full-length APPSw. Both inhibited intracellular formation of the beta-cleaved product. These results suggest that peptide aldehydes such as MG132 have multiple effects on the maturation and processing of APP. We conclude that the MG132-induced decrease in beta-secretase cleavage of APPSw is due to a block in maturation. This is sufficient to explain the previously reported peptide aldehyde-induced decrease in Abeta peptide secretion.

Elevated levels of protein-bound p-hydroxyphenylacetaldehyde, an amino-acid-derived aldehyde generated by myeloperoxidase, are present in human fatty streaks, intermediate lesions and advanced atherosclerotic lesions.

Reactive aldehydes might have a pivotal role in the pathogenesis of atherosclerosis by covalently modifying low-density lipoprotein (LDL). However, the identities of the aldehyde adducts that form on LDL in vivo are not yet clearly established. We previously demonstrated that the haem protein myeloperoxidase oxidizes proteins in the human artery wall. We also have shown that p-hydroxyphenylacetaldehyde (pHA), the aldehyde that forms when myeloperoxidase oxidizes L-tyrosine, covalently modifies the N(epsilon)-lysine residues of proteins. The resulting Schiff base can be quantified as N(epsilon)-[2-(p-hydroxyphenyl)ethyl]lysine (pHA-lysine) after reduction with NaCNBH(3). Here we demonstrate that pHA-lysine is a marker for LDL that has been modified by myeloperoxidase, and that water-soluble, but not lipid-soluble, antioxidants inhibit the modification of LDL protein. To determine whether myeloperoxidase-generated aldehydes might modify LDL in vivo, we used a combination of isotope-dilution GC-MS to quantify pHA-lysine in aortic tissues at various stages of lesion evolution. We also analysed LDL isolated from atherosclerotic aortic tissue. Comparison of normal and atherosclerotic aortic tissue demonstrated a significant elevation (more than 10-fold) of the reduced Schiff base adduct in fatty streaks, intermediate lesions and advanced lesions compared with normal aortic tissue. Moreover, the level of pHA-lysine in LDL recovered from atherosclerotic aortic intima was 200-fold that in plasma LDL of healthy donors. These results indicate that pHA-lysine, a specific covalent modification of LDL, is generated in human atherosclerotic vascular tissue. They also raise the possibility that reactive aldehydes generated by myeloperoxidase have a role in converting LDL into an atherogenic lipoprotein.

Electron paramagnetic resonance detection of free tyrosyl radical generated by myeloperoxidase, lactoperoxidase, and horseradish peroxidase.

Phagocytes secrete the heme protein myeloperoxidase, which is present and active in human atherosclerotic tissue. These cells also generate hydrogen peroxide (H2O2), thereby allowing myeloperoxidase to generate a range of oxidizing intermediates and stable end products. When this system acts on L-tyrosine in vitro, it forms o, o'-dityrosine, which is enriched in atherosclerotic lesions. Myeloperoxidase, therefore, may oxidize artery wall proteins in vivo, cross-linking their L-tyrosine residues. In these studies, we used electron paramagnetic resonance (EPR) spectroscopy to identify an oxidizing intermediate in this reaction pathway and in parallel reactions catalyzed by horseradish peroxidase and lactoperoxidase. Using an EPR flow system to rapidly mix and examine solutions containing horseradish peroxidase, H2O2, and L-tyrosine, we detected free tyrosyl radical (a2,6H = 6.3 G, a3,5H = 1.6 G, and abetaH = 15. 0 G). We then used spin trapping techniques with 2-methyl-2-nitrosopropane (MNP) to further identify this intermediate. The resulting three-line spectrum (aN = 15.6 G) was consistent with an MNP/tyrosyl radical spin adduct. Additional MNP spin trapping studies with ring-labeled L-[13C6]tyrosine yielded a characteristic eight-line EPR spectrum (aN = 15.6 G, a13C (2) = 8.0 G, a13C (1) = 7.1 G, a13C (1) = 1.3 G), indicating that the MNP adduct resulted from trapping a carbon-centered radical located on the aromatic ring of L-tyrosine. This same eight-line spectrum was observed when human myeloperoxidase or bovine lactoperoxidase was substituted for horseradish peroxidase. Furthermore, a partially immobilized MNP/tyrosyl radical spin adduct was detected when we exposed a synthetic polypeptide composed of glutamate and L-tyrosine residues to the myeloperoxidase-H2O2-L-tyrosine system. The broadened EPR signal resulting from this MNP/polypeptide adduct was greatly narrowed by proteolytic digestion with Pronase, confirming that the initial spin-trapped radical was protein-bound. Collectively, these results indicate that peroxidases use H2O2 to convert L-tyrosine to free tyrosyl radical. They also support the idea that free tyrosyl radical initiates cross-linking of L-tyrosine residues in proteins. We suggest that this pathway may play an important role in protein and lipid oxidation at sites of inflammation and in atherosclerotic lesions.

The chaperone BiP/GRP78 binds to amyloid precursor protein and decreases Abeta40 and Abeta42 secretion.

Recent studies of cellular amyloid precursor protein (APP) metabolism demonstrate a beta-/gamma-secretase pathway resident to the endoplasmic reticulum (ER)/Golgi resulting in intracellular generation of soluble APP (APPsbeta) and Abeta42 peptide. Thus, these intracellular compartments may be key sites of amyloidogenic APP metabolism and Alzheimer's disease pathogenesis. We hypothesized that the ER chaperone immunoglobulin binding protein (BiP/GRP78) binds to and facilitates correct folding of nascent APP. Metabolic labeling and immunoprecipitation of transiently transfected human embryonic kidney 293 cells demonstrated co-precipitation of APP with GRP78, revealing their transient interaction in the ER. Maturation of cellular APP was impaired by this interaction. Furthermore, the levels of APPs, Abeta40, and Abeta42 recovered in conditioned medium were lower compared with cells transfected with APP alone. Co-expression with APP of GRP78 T37G, an ATPase mutant, almost completely blocked cellular APP maturation as well as recovery of APPs, Abeta40, and Abeta42 in conditioned medium. The inhibitory effects of GRP78 and GRP78 T37G on Abeta40 and Abeta42 secretion were magnified by co-expression with the Swedish mutation of APP (K670N/M671L). Collectively, these data suggest a transient and direct interaction of GRP78 with APP in the ER that modulates intracellular APP maturation and processing and may facilitate its correct folding.

Slo3, a novel pH-sensitive K+ channel from mammalian spermatocytes.

Potassium channels have evolved to play specialized roles in both excitable and inexcitable tissues. Here we describe the cloning and expression of Slo3, a novel potassium channel abundantly expressed in mammalian spermatocytes. Slo3 represents a new and unique type of potassium channel regulated by both intracellular pH and membrane voltage. Reverse transcription-polymerase chain reaction, Northern analysis, and in situ hybridization show that Slo3 is primarily expressed in testis in both mouse and human. Because of its sensitivity to both pH and voltage, Slo3 could be involved in sperm capacitation and/or the acrosome reaction, essential steps in fertilization where changes in both intracellular pH and membrane potential are known to occur. The protein sequence of mSlo3 (the mouse Slo3 homologue) is similar to Slo1, the large conductance, calcium- and voltage-gated potassium channel. These results suggest that Slo channels comprise a multigene family, defined by a combination of sensitivity to voltage and a variety of intracellular factors. Northern analysis from human testis indicates that a Slo3 homologue is present in humans and conserved with regard to sequence, transcript size, and tissue distribution. Because of its high testis-specific expression, pharmacological agents that target human Slo3 channels may be useful in both the study of fertilization as well as in the control or enhancement of fertility.

p-Hydroxyphenylacetaldehyde, the major product of L-tyrosine oxidation by the myeloperoxidase-H2O2-chloride system of phagocytes, covalently modifies epsilon-amino groups of protein lysine residues.

Activated human phagocytes employ the myeloperoxidase-H2O2-Cl- system to convert L-tyrosine to p-hydroxyphenylacetaldehyde (pHA). We have explored the possibility that pHA covalently reacts with proteins to form Schiff base adducts, which may play a role in modifying targets at sites of inflammation. Because Schiff bases are labile to acid hydrolysis, prior to analysis the adducts were rendered stable by reduction with NaCNBH3. Purified pHA reacted with Nalpha-acetyllysine, an analog of protein lysine residues. The reduced reaction product was identified as Nalpha-acetyl-Nepsilon-(2-(p-hydroxyphenyl)ethyl)lysine by 1H NMR spectroscopy and mass spectrometry. The compound Nepsilon-(2-(p-hydroxyphenyl)ethyl)lysine (pHA-lysine) was likewise identified in acid hydrolysates of bovine serum albumin (BSA) that were first exposed to myeloperoxidase, H2O2, L-tyrosine, and Cl- and then reduced with NaCNBH3. Other halides (F-, Br-, I-) and the pseudohalide SCN- could not replace Cl- as a substrate in the myeloperoxidase-H2O2-L-tyrosine system. In the absence of the enzymatic system, pHA-lysine was detected in reduced reaction mixtures of BSA, L-tyrosine, and reagent HOCl. In contrast, pHA-lysine was undetectable when BSA was incubated with L-tyrosine and HOBr, peroxynitrite, hydroxyl radical, or a variety of other peroxidases, indicating that the aldehyde-protein adduct was selectively produced by HOCl. Human neutrophils activated in the presence of tyrosine also modified BSA lysine residues. pHA-lysine formation required L-tyrosine and cell activation; it was inhibited by peroxidase inhibitors and catalase, implicating myeloperoxidase and H2O2 in the reaction pathway. pHA-lysine was detected in inflamed human tissues that were reduced, hydrolyzed, and then analyzed by mass spectrometry, indicating that the reaction of pHA with proteins may be of physiological importance. These observations raise the possibility that the identification of pHA-lysine in tissues will pinpoint targets where phagocytes inflict oxidative damage in vivo.

In vivo threonine phosphorylation of immunoglobulin binding protein (BiP) maps to its protein binding domain.

Immunoglobin binding protein (BiP) molecules exist as both monomers and oligomers and phosphorylated BiP is restricted to the oligomeric pool. Modified BiP is not bound to proteins such as immunoglobulin heavy chain and consequently, may constitute an inactive form. Unlike earlier analysis of mammalian BiP isolated by two-dimensional gel electrophoresis, results here demonstrated that immunoprecipitated BiP displayed predominantly threonine phosphorylation with only a trace of detectable phosphoserine. Like other Hsp70 family members, BiP is comprised of three domains: an amino terminal domain which binds nucleotide, an 18 kilodalton domain which binds peptide, and a carboxyl terminal variable domain of unknown function. Cyanogen bromide cleavage and enzymatic digestion experiments mapped threonine phosphorylation to a site within a 47 amino acid sequence of the peptide binding domain which contains seven threonine residues. Partial proteinase K digestion in the presence of ATP independently verified that the in vivo phosphorylation site of mammalian (BiP) is located within the peptide binding domain. Furthermore, phosphorylation did not impede BiPs ATP-induced conformational change. Thus, the peptide binding domain of BiP is phosphorylated on threonine residue(s) mapping to not more than two tryptic fragments within the peptide binding domain. This location on the molecule could explain why phosphorylated BiP is not detected bound to proteins in vivo.

Inhibition of immunoglobulin folding and secretion by dominant negative BiP ATPase mutants.

A group of resident ER proteins have been identified that are proposed to function as molecular chaperones. The best characterized of these is BiP/GRP78, an hsp70 homologue that binds peptides containing hydrophobic residues in vitro and unfolded or unassembled proteins in vivo. However, evidence that mammalian BiP plays a direct role in protein folding remains circumstantial. In this study, we examine how BiP interacts with a particular substrate, immunoglobulin light chain (lambda LC), during its folding. Wild-type hamster BiP and several well-characterized BiP ATPase mutants were used in transient expression experiments. We demonstrate that wild-type lambda LCs showed prolonged association with mutant BiP which inhibited their secretion. Both wild-type and mutant BiP bound only to unfolded and partially folded LCs. The wild-type BiP was released from the incompletely folded LCs, allowing them to fold and be secreted, whereas the mutant BiP was not released. As a result, the LCs that were bound to BiP mutants were unable to undergo complete disulfide bond formation and were retained in the ER. Our experiments suggest that LCs undergo both BiP-dependent and BiP-independent folding steps, demonstrating that both ATP binding and hydrolysis activities of BiP are essential for the completion of LC folding in vivo and reveal that BiP must release before disulfide bond formation can occur in that domain.

In vitro dissociation of BiP-peptide complexes requires a conformational change in BiP after ATP binding but does not require ATP hydrolysis.

In the present study, we produced single point mutations in the ATP binding site of hamster BiP, isolated recombinant proteins, and characterized them in terms of their affinity for ATP and ADP, their ability to undergo a conformational change upon nucleotide binding, and their rate of ATP hydrolysis. These analyses allowed us to classify the mutants into three groups: ATP hydrolysis (T229G), ATP binding (G226D, G227D), and ATP-induced conformation (T37G) mutants, and to test the role of these activities in the in vitro ATP-mediated release of proteins from BiP. All three classes of mutants were still able to bind peptide demonstrating that nucleotide is not involved in this function. Addition of ATP to either wild-type BiP or the T229G mutant caused the in vitro release of bound peptide, confirming that ATP hydrolysis is not required for protein release. ATP did not dissociate G226D, G227D, or T37G mutant BiP-peptide complexes, suggesting that ATP binding to BiP is not sufficient for the release of bound peptides, but that an ATP-induced conformational change in BiP is necessary. The identification of BiP mutants that are defective in each of these steps of ATP hydrolysis will allow the in vivo dissection of the role of nucleotide in BiP's activity.

In vivo expression of mammalian BiP ATPase mutants causes disruption of the endoplasmic reticulum.

BiP possesses ATP binding/hydrolysis activities that are thought to be essential for its ability to chaperone protein folding and assembly in the endoplasmic reticulum (ER). We have produced a series of point mutations in a hamster BiP clone that inhibit ATPase activity and have generated a species-specific anti-BiP antibody to monitor the effects of mutant hamster BiP expression in COS monkey cells. The enzymatic inactivation of BiP did not interfere with its ability to bind to Ig heavy chains in vivo but did inhibit ATP-mediated release of heavy chains in vitro. Immunofluorescence staining and electron microscopy revealed vesiculation of the ER membranes in COS cells expressing BiP ATPase mutants. ER disruption was not observed when a "44K" fragment of BiP that did not include the protein binding domain was similarly mutated but was observed when the protein binding region of BiP was expressed without an ATP binding domain. This suggests that BiP binding to target proteins as an inactive chaperone is responsible for the ER disruption. This is the first report on the in vivo expression of mammalian BiP mutants and is demonstration that in vitro-identified ATPase mutants behave as dominant negative mutants when expressed in vivo.

The modification and assembly of proteins in the endoplasmic reticulum.

Proteins fold and assemble in the endoplasmic reticulum in an environment that is very different from the cytosol. The presence of relatively high concentrations of calcium, an oxidizing state, ATP and lumenal proteins are all important in mediating these events.

The immunoglobulin-binding protein in vitro autophosphorylation site maps to a threonine within the ATP binding cleft but is not a detectable site of in vivo phosphorylation.

In vitro incubation of immunoprecipitated immunoglobulin-binding protein (BiP) complexes with calcium and [gamma-32P]ATP resulted in the phosphorylation of BiP on a threonine residue. This autophosphorylation activity did not occur in the presence of magnesium but had the same pH optimum as reported for its magnesium-dependent ATPase activity. This suggested the possibility that both activities could occur through ATP hydrolysis at the same site. In support of this, mutation of either Thr-37 or Thr-229 to a glycine eliminated both autophosphorylation and ATPase activities, and mutation of either residue to a serine significantly reduced both activities. Glutamic acid 175 in HSC71 has been hypothesized to flank the divalent cation complexed with ATP. Mutation of the analogous glutamic acid, Glu-201, in BiP abolished ATPase activity but still supported some autophosphorylation. The in vitro phosphorylation site was mapped to Thr-229 by mutational analysis. This threonine has been hypothesized to interact with the gamma-phosphate of ATP through a polarized water molecule and would be in a position to act as a phosphate acceptor in the ATP hydrolysis reaction. These data imply that both ATPase and autophosphorylation result from ATP hydrolysis at the same site and that the cation associated with BiP determines which activity is observed. Comparison of partial protease digestion or cyanogen bromide cleavage products of in vitro and in vivo phosphorylated BiP demonstrated that Thr-229 is not a detectable site of phosphorylation in cells. Therefore, whatever functional role phosphorylation may have in vivo, it cannot be attributed to autophosphorylation of Thr-229.

Mutations within the nucleotide binding site of immunoglobulin-binding protein inhibit ATPase activity and interfere with release of immunoglobulin heavy chain.

Immunoglobulin-binding protein (BiP), a 70-kDa heat shock protein in the endoplasmic reticulum, binds transiently to nascent proteins, releasing them upon folding and assembly. The in vitro release of bound proteins from BiP requires ATP hydrolysis. Recently, the three-dimensional structure was solved for an ATP-hydrolyzing proteolytic 44-kDa fragment of a 71-kDa heat shock cognate protein, HSC71. Because of the high degree of homology in this region, BiP presumably forms a similar ATP binding structure. Amino-terminal deletions in BiP eliminated ATP-agarose binding. Alteration of a second potential ATP binding site had no effect, suggesting that only the HSC71-like site was capable of ATP binding. Crystallographic data from HSC71 implicated certain amino acids in interactions with the beta-phosphate, gamma-phosphate, and divalent cation of ATP. Mutation of each corresponding residue in BiP (Thr-37, Thr-229, and Glu-201) severely inhibited its ATPase activity. These BiP mutants were still capable of binding ATP and immunoglobulin heavy chains, suggesting that these mutations did not drastically alter the structure of BiP. They did however block the ATP-mediated release of heavy chains from BiP. Our results demonstrate that the structure of BiP in this region must be extremely similar to that elucidated for HSC71 and that mutations of residues proposed to interact with ATP block the ATP-mediated release of bound protein by inhibiting ATP hydrolysis.