Presenilin-1 mutations downregulate the signalling pathway of the unfolded-protein response.

Missense mutations in the human presenilin-1 (PS1) gene, which is found on chromosome 14, cause early-onset familial Alzheimer's disease (FAD). FAD-linked PS1 variants alter proteolytic processing of the amyloid precursor protein and cause an increase in vulnerability to apoptosis induced by various cell stresses. However, the mechanisms responsible for these phenomena are not clear. Here we report that mutations in PS1 affect the unfolded-protein response (UPR), which responds to the increased amount of unfolded proteins that accumulate in the endoplasmic reticulum (ER) under conditions that cause ER stress. PS1 mutations also lead to decreased expression of GRP78/Bip, a molecular chaperone, present in the ER, that can enable protein folding. Interestingly, GRP78 levels are reduced in the brains of Alzheimer's disease patients. The downregulation of UPR signalling by PS1 mutations is caused by disturbed function of IRE1, which is the proximal sensor of conditions in the ER lumen. Overexpression of GRP78 in neuroblastoma cells bearing PS1 mutants almost completely restores resistance to ER stress to the level of cells expressing wild-type PS1. These results show that mutations in PS1 may increase vulnerability to ER stress by altering the UPR signalling pathway.

Increased serum levels of S100A12 in patients with MPO-ANCA-associated glomerulonephritis.

BACKGROUND: Increased serum levels of S100A12, a proinflammatory protein secreted by activated neutrophils, have recently been shown in patients with active inflammatory diseases, such as rheumatoid arthritis and Kawasaki disease. In this study, we investigated serum levels of S100A 12 in patients with small-vessel vasculitis, myeloperoxidase anti-neutrophil cytoplasmic antibodies- (MPO-ANCA) associated pauci-immune glomerulonephritis. METHODS: Serum S100A12 concentrations were measured by a sandwich enzyme-linked immunosorbent assay (ELISA) in 46 patients with MPO-ANCA-associated glomerulonephritis and 29 healthy controls. We analyzed correlations between serum S100A12 levels and a clinical index of vasculitis activity, the Birmingham Vasculitis Activity Score (BVAS), various laboratory parameters, and pathological activity scores in the patients. We also analyzed changes of serum S100A12 levels in 10 patients after treatment. RESULTS: ELISA showed about 4-fold higher levels of serum S100A12 in patients with MPO-ANCA-associated glomerulonephritis than healthy controls. Serum S100A12 levels correlated with the BVAS scores, the peripheral white blood cell count, levels of serum C-reactive protein and creatinine, and pathological activity scores in the patients, but did not correlate with serum MPO-ANCA titers. Serum S100A12 levels after treatment decreased in all the 10 patients examined. CONCLUSION: We demonstrated that increased serum S100A12 levels correlate with clinical, laboratory and pathological parameters of disease activity in patients with MPO-ANCA-associated glomerulonephritis. Serum S100A12 level may be one of the useful markers of disease activity in MPO-ANCA-associated glomerulonephritis.

Molecular cloning of a novel membrane glycoprotein, pal, specifically expressed in photoreceptor cells of the retina and containing leucine-rich repeat.

We have isolated a novel retina-specific gene in a screen for genes of which expression is not apparent neonatally in rat retina but is abundant postnatally on day 14 (P14). This gene, named Pal, encodes a putative type I transmembrane protein containing five leucine-rich repeats (LRRs), a single C2-type Ig-like domain, and a single fibronectin type III domain and is considered to be a new member of the LRR and Ig superfamily. No expression of Pal was found in rat retina at P1, but it was detected at P7 and markedly increased with subsequent development. These expression patterns of Pal appeared to be correlated with the development of the photoreceptor outer segments, because in the adult rat retina it was specifically localized in these segments. Ultrastructually, Pal immunoreactivity was distributed diffusely on the disk membrane in the lamellar regions. On the basis of its structural features and localization pattern, Pal may act as a receptor for a certain trophic factor or for an adhesion molecule participating in morphogenesis. The human homolog of Pal was mapped to chromosome 10q23.2-23.3 using fluorescence in situ hybridization.

Induced HMGA1a expression causes aberrant splicing of Presenilin-2 pre-mRNA in sporadic Alzheimer's disease.

The aberrant splicing isoform (PS2V), generated by exon 5 skipping of the Presenilin-2 (PS2) gene transcript, is a diagnostic feature of sporadic Alzheimer's disease (AD). We found PS2V is hypoxia-inducible in human neuroblastoma SK-N-SH cells. We purified a responsible trans-acting factor based on its binding to an exon 5 fragment. The factor was identified as the high mobility group A1a protein (HMGA1a; formerly HMG-I). HMGA1a bound to a specific sequence on exon 5, located upstream of the 5' splice site. HMGA1a expression was induced by hypoxia and the protein was accumulated in the nuclear speckles with the endogenous splicing factor SC35. Overexpression of HMGA1a generated PS2V, but PS2V was repressed by cotransfection with the U1 snRNP 70K protein that has a strong affinity to HMGA1a. HMGA1a could interfere with U1 snRNP binding to the 5' splice site and caused exon 5 skipping. HMGA1a levels were significantly increased in the brain tissue from sporadic AD patients. We propose a novel mechanism of sporadic AD that involves HMGA1a-induced aberrant splicing of PS2 pre-mRNA in the absence of any mutations.

Carotid plaque characterization using 3D T1-weighted MR imaging with histopathologic validation: a comparison with 2D technique.

BACKGROUND AND PURPOSE: 3D FSE T1WI has recently been used for carotid plaque imaging, given the potential advantages in contrast and spatial resolutions. However, its diagnostic performance remains unclear. Hence, we compared the ability of this technique to readily assess plaque characteristics with that of conventional images and validated the results with histologic classification. MATERIALS AND METHODS: We prospectively examined 34 patients with carotid stenosis who underwent carotid endarterectomy by using 1.5T scanners and obtained 3D-FSE T1WI and 2D spin-echo T1WI scans. After generating reformatted images obtained from the 3D-FSE T1-weighted images, we calculated the contrast ratios for the plaques and the adjacent muscles and compared these findings with the pathologic classifications. RESULTS: Carotid plaques were histologically classified as types VII, VIII, IV-V, or VI. With 3D-FSE T1WI, the range of contrast ratios for each classification was the following: 0.94-0.97 (median, 0.95), 0.95-1.29 (median, 1.10), 1.33-1.54 (median, 1.42), and 1.53-2.12 (median, 1.80), respectively. With 2D imaging, the range of contrast ratios for each classification was the following: 0.79-1.02 (median, 0.90), 0.88-1.19 (median, 1.01), 1.17-1.46 (median, 1.23), and 1.55-2.51 (median, 2.07), respectively. Results were significantly different among the 4 groups (P < .001). Sensitivity and specificity for discriminating vulnerable plaques (IV-VI) from stable plaques (VII, VIII) were both 100% for the 3D technique and 100% and 91%, respectively, for the 2D technique. CONCLUSIONS: 3D-FSE T1WI accurately characterizes intraplaque components of the carotid artery, with excellent sensitivity and specificity compared with those of 2D-T1WI.

Mutations of p53 in gallbladder carcinomas in high-incidence areas of Japan and Chile.

Gallbladder adenocarcinomas from patients in two high-prevalence areas, Niigata (Japan) and Santiago (Chile), were analyzed for acquired mutations in exons 5-8 of the p53 tumor suppressor gene, and the characteristics of p53 alterations in the two groups were compared. Of 42 tumors, 22 (52.4%) harbored 25 alterations identified by PCR amplification and direct sequencing (13 of 22 tumors from Niigata and 12 of 20 tumors from Santiago). All alterations were single base pair substitutions, 20 (80%) leading to an amino acid substitution or a chain-termination signal, and 5 (20%) were silent. Immunohistochemically, 55 of 84 cases (65.5%) showed overexpression of p53 protein, with no significant difference in frequency between the two areas. Missense mutations correlated highly with overexpression of the p53 protein (93.4%). Mutations of p53 occurred in all four exons examined, most commonly in exon 5, but in no particular "hot spot." In base-change spectra, all 12 mutations from Santiago showed transitions, with 4 arising at the CpG dinucleotide (33.3%). In contrast, no such transition was found at CpG sites in Niigata, and 4 of 13 mutations (30.8%) were transversions. The data indicated that p53 mutations are highly important in carcinogenesis in the gallbladder. In addition, the difference in p53 mutational spectra in Niigata and Santiago indicate a likely regional difference in mutagenesis.

Characterization of mouse Ire1 alpha: cloning, mRNA localization in the brain and functional analysis in a neural cell line.

In yeast, an endoplasmic reticulum (ER)-associated protein, Ire1p, is believed to initiate the unfolded protein response (UPR), that is responsible for protein folding in the ER under stressed conditions. Two mammalian homologs of Ire1p have been identified, Ire1 alpha and Ire1 beta. We have previously reported that familial Alzheimer's disease linked presenilin-1 variants downregulate the signaling pathway of the UPR by affecting the phosphorylation of Ire1 alpha. In the present study, we cloned the mouse homolog of Ire1 alpha for generating genetically modified mice. Ire1 alpha was ubiquitously expressed in all mouse tissues examined, and was expressed preferentially in neuronal cells in mouse brain. This led us to investigate the effects of the downregulation of the UPR on the survival of neuronal cells under conditions of ER stress. Morphological and biochemical studies using a dominant-negative form of mouse Ire1 alpha have revealed that cell death caused by ER stress can be attributed to apoptosis, and that the downregulation of the UPR enhances the apoptotic process in the mouse neuroblastoma cell line, Neuro2a. Our results indicate that genetically modified mice such as transgenic mice with a dominant-negative form of Ire1 alpha might provide further understanding of the pathogenic mechanisms of Alzheimer's disease and other neurodegenerative disorders.

Absence of endoproteolysis but no effects on amyloid beta production by alternative splicing forms of presenilin-1, which lack exon 8 and replace D257A.

It is well known that presenilin-1 (PS1) is involved in cleavage of amyloid precursor protein (APP) at the gamma-secretase site, and that the amino acids residues of D257 and D385 in PS1 are critical for this cleavage of APP and the endoproteolysis of itself. An alternatively spliced form of PS1 skipping exon 8 (PS1d8), which has D257A at the splice junction of exon 7/9, is expressed in human brain and in some cell lines. In this study, we examined production of Amyloid beta (A beta) and the endoproteolysis of the holoproteins in PS1d8-expressing neuroblastoma cells. Western blotting showed an absence of endoproteolysis in PS1d8. However, PS1d8 did not affect the production of A beta, which is different from the artificial point mutant PS1D257A. These results suggest that endoproteolysis of PS1 and gamma-secretase activity could be independent.

Purification and characterization of 1,2-alpha-mannosidase of Aspergillus oryzae.

1,2-alpha-Mannosidase was purified approximately 1,400-fold from an enzyme product of Aspergillus oryzae. The enzyme showed a single band in disc gel electrophoresis and the molecular weight was estimated to be about 49,000 daltons by gel exclusion chromatography. The substrate specificity of the enzyme was examined with mannooligosaccharides, yeast mannan, glycopeptides, and a glycoprotein. The alpha-(1 leads to 2)-linking mannose residues located at the nonreducing-ends of the substrates were selectively removed by the enzyme, whereas p-nitrophenyl alpha-D-mannopyranoside was completely stable to the enzyme. alpha-(1 leads to 2)-Linking mannose residues in intact bovine pancreatic ribonuclease B were also removed completely with the enzyme. The enzyme showed an optimum pH in the range of pH 4.9 to 5.3 and had a Km value of 0.57 mM with 1,2-alpha-mannobiose. The present alpha-mannosidase was quite stable, and the activity was inhibited by D-mannono-gamma-lactone and by heavy metal ions, including zinc ions.

Purification and characterization of endo-beta-N-acetylglucosaminidase of Aspergillus oryzae.

An endo-beta-N-acetylglucosaminidase which hydrolyzes the N,N'-diacetylchitobiosyl linkage in asparagine-linked oligosaccharides was purified from the enzyme product of Aspergillus oryzae. Its substrate specificity was similar to that of endo-beta-N-acetylglucosaminidase H from Streptomyces griseus with respect to the relative activities toward the glycopeptides obtained from ovalbumin and bovine IgG. The present endoglycosidase exhibited a broad optimum pH range and was relatively stable. Metal ions, chelating agents and D-mannose did not have a significant effect on the enzyme activity.

Substrate-dependent change in the pH-activity profile of alkaline endo-1,4-beta-glucanase from an alkaline Bacillus sp.

A neutral cellulase (BSC) from Bacillus subtilis and an alkaline cellulase (NK1) from alkalophilic Bacillus sp. N-4 show significant amino acid sequence homology. Despite the high homology, the pH-activity profiles of the two enzymes for carboxymethyl cellulose (CMC) hydrolysis are quite different; BSC shows a sharp optimum pH at 6, whereas NK1 shows its full activity in a broad range, from pH 6 to 10.5. For elucidation of the reasons for the difference in their pH-activity profiles, their activities were examined at various pHs using a series of cellooligosaccharides and their derivatives, cellotetraose (G4), cellopentaose (G5), cellohexaose (G6), cellopentaitol (G5OH), and cellohexaitol (G6OH), as substrates. The optimum pH of BSC was around 6 for all the cellooligosaccharides examined. On the other hand, the optimum pH of NK1 varied depending on the substrate, i.e., a sharp optimum at pH 6 with G4 and G5OH, and a broad optimum of pH 6 to 10.5 with G5, G6, and G6OH. Comparison of the kinetic parameters of the two cellulases at pH 7 and 9 using G6OH as a substrate revealed that NK1 showed similar values at both pHs, while BSC showed a greatly increased Km value for this substrate at pH 9. In addition, NK1 showed a greatly increased Km value for G5OH hydrolysis at pH 9. Both enzymes cleaved these substrates at the same position, which suggests the same productive binding mode of these substrates with both enzymes. All these observations suggest that the reduced enzyme activity of BSC in the alkaline pH range can be attributed to a decrease in the affinity of a subsite for the third glucose moiety from the scissile site of these substrates.

Correlation of p53 protein expression with apoptotic incidence in colorectal neoplasia.

The wild-type p53 gene suppresses cell proliferation and induces apoptosis when it is transfected into human colon cancer cell lines. Therefore, mutation of the p53 gene, which correlates closely with p53 protein overexpression, would be predicted to activate cell proliferation and limit apoptosis. We tested this hypothesis by correlating p53 protein expression with cell proliferation and apoptosis in 70 neoplasms (29 adenomas and 41 carcinomas) using p53 and Ki-67 immunohistochemical staining and DNA nick end labelling. The p53 immunoreactivity was independent of the Ki-67 positivity. The apoptotic incidence was less frequent (P < 0.005) in tumours with diffuse p53 protein overexpression than in those with the sporadic overexpression, defined as p53 staining of isolated or scattered expression. In addition, apoptotic incidence only correlated directly (P < 0.05) with Ki-67 positivity in tumours with sporadic p53-protein expression. These results indicate that p53 protein that is expressed sporadically in colorectal neoplasms is probably wild-type protein and induces apoptosis in response to active cell proliferation. In contrast, diffusely overexpressed p53 protein in colorectal neoplasms is probably mutant and correlates with a reduction in apoptotic cell death independently of cell proliferation.

[Observation of human corneal and scleral collagen fibrils by atomic force microscopy].

PURPOSE: We attempted to analyze the three-dimensional ultrastructure of human corneal and scleral collagen fibrils with an atomic force microscope (AFM). METHODS: A normal eye removed from a 66-year-old male was used in the study. Suspended corneal and scleral collagen fibrils were individually attached to glass slides by centrifugation. These collagen fibrils were air-dried and observed with a non-contact mode AFM in air. RESULTS: AFM imaging provided information on the surface topography of both corneal and scleral collagen fibrils. The corneal collagen fibrils had a height of 11.9 +/- 1.0 (mean +/- standard deviation) nm and the scleral fibrils of 82.5 +/- 35.6 nm. A periodic banding pattern of grooves and ridges was clearly found in both types of fibrils; the D-periodicity and the groove depth were 65.7 +/- 0.8 nm and 1.46 +/- 0.50 nm in the corneal fibrils, and 67.3 +/- 1.1 nm and 6.16 +/- 1.23 nm in the scleral fibrils. CONCLUSIONS: Surface topographic images of human corneal and scleral collagen fibrils were clearly obtained with the AFM. This technique provides quantitative information on the surface morphology of the collagen fibrils at high resolution.

Predicting carotid plaque characteristics using quantitative color-coded T1-weighted MR plaque imaging: correlation with carotid endarterectomy specimens.

BACKGROUND AND PURPOSE: MR plaque imaging is used to evaluate the risk of embolic complications during carotid endarterectomy and carotid artery stent placement. However, its performance for characterizing intraplaque components has varied across studies and is generally suboptimal. Hence, we correlated MR imaging results with histologic findings to determine whether a combination of high-contrast T1-weighted imaging and quantitative image analysis could readily determine plaque characteristics. MATERIALS AND METHODS: We prospectively examined 40 consecutive patients before carotid endarterectomy by using a 1.5T scanner and axial T1-weighted spin-echo images under optimized scanning conditions. The percentage areas of intraplaque fibrous tissue, lipid/necrosis, and hemorrhage were calculated automatically by using the software with previously reported cutoff values and were compared with those of the specimens. The thickness of the fibrous cap was also measured manually. RESULTS: The percentage areas of fibrous, lipid/necrotic, and hemorrhagic components were 5.7%-98.7%, 1.3%-65.7%, and 0%-82.0%, respectively, as determined by the MR images, whereas the corresponding values were 4.8%-92.3%, 7.0%-93.8%, and 0%-70.4%, respectively, as determined by histologic examination. Significant positive correlation and agreement were observed between MR images and histologic specimens (r = 0.92, 0.79, and 0.92; intraclass correlation coefficients = 0.91, 0.67, and 0.89; respectively). Thickness of the fibrous caps on MR images (0.21-0.87 mm) and in the specimens (0.14-0.83 mm) also showed positive correlation and agreement (r = 0.61, intraclass correlation coefficient = 0.59). CONCLUSIONS: Quantitative analysis of high-contrast T1-weighted images can accurately evaluate the composition of carotid plaques in carotid endarterectomy candidates.

Prediction of carotid plaque characteristics using non-gated MR imaging: correlation with endarterectomy specimens.

BACKGROUND AND PURPOSE: Electrocardiographic gating, commonly used in MR carotid plaque imaging, can negatively affect intraplaque contrast if the TR is inappropriate. The present study aimed to determine whether a non-gated technique with appropriate TRs can accurately evaluate intraplaque characteristics in specimens excised by CEA. MATERIALS AND METHODS: We prospectively examined 40 consecutive patients who underwent CEA (59-82 years of age) by using a 1.5T scanner. Axial T1WI with a TR of 500 ms and PDWI and T2WI with a TR of 3000 ms with a self-navigated rotating-blade scan instead of cardiac gating were obtained. Signal intensities of the plaque and adjacent muscle were measured, and the CR on T1WI, PDWI, and T2WI as well as the gray-scale median on US were correlated with the pathologic findings of the CEA specimens. RESULTS: On T1WI, the CRs of the carotid plaques differed significantly among groups in which the main components were histologically confirmed as fibrous tissue, lipid/necrosis, and hemorrhage (0.54-1.17, 1.16-1.53, and 1.40-2.29, respectively). The sensitivity and specificity for discriminating lipid/necrosis/hemorrhage from fibrous tissue were 96% and 100%, respectively. On T2WI, the CRs of plaques with lipid/necrosis were significantly higher than those of other groups, but the CRs on PDWI and the gray-scale median on US were not significantly different among the groups. CONCLUSIONS: Non-gated MR plaque imaging, particularly T1WI, can readily predict the intraplaque main components of the carotid artery with high sensitivity and specificity.

A novel role for RIP1 kinase in mediating TNFα production.

Receptor-interacting protein 1 (RIP1) is a Ser/Thr kinase with both kinase-dependent and kinase-independent roles in death receptor signaling. The kinase activity of RIP1 is required for necroptosis, a caspase-independent pathway of programmed cell death. In some cell types, the inhibition of caspases leads to autocrine production of TNFα, which then activates necroptosis. Here, we describe a novel role for RIP1 kinase in regulating TNFα production after caspase inhibition. Caspase inhibitors activate RIP1 kinase and another protein, EDD, to mediate JNK signaling, which stimulates Sp1-dependent transcription of TNFα. This pathway is independent of nuclear factor κB and also occurs after Smac mimetic/IAP antagonist treatment or the loss of TNF receptor-associated factor 2 (Traf2). These findings implicate cIAP1/2 and Traf2 as negative regulators of this RIP1 kinase-dependent TNFα production pathway and suggest a novel role for RIP1 kinase in mediating TNFα production under certain conditions.

The terminal insertional segments and communications of the vertebral nerve in the human cervical region.

Twenty-four cadavers (48 sides) were used to clarify the terminal insertional segment and communications of the vertebral nerve in the cervical region under a surgical microscope. After displacing the prevertebral muscles (longus colli and longus capitis) laterally, the ventral parts of the transverse foramen of vertebrae (from C2 to C6) were removed, and the insertional segment and communicates of the vertebral nerve surrounding the vertebral artery were observed. The results showed: (1) the vertebral nerve ascended along the ventral or mediodorsal vertebral artery and terminated mainly at C3 (22/36 sides) but not terminated at C4 or C5 only; (2) the superficial communicates from the cervical sympathetic trunk ran in a proximal and distal direction when the fibers entered the anterior branches of the cervical nerves. The fibers running to the proximal direction communicated with the vertebral nerve in the part of transverse foramen; (3) motor and/or sensory rami supplying the prevertebral muscles, corpus vertebrae and intervertebral discs could pass through an "arched-shaped" fiber bundle on the ventral surface of the vertebral artery. In conclusion, the vertebral nerve and the fibers surrounding the vertebral artery could be considered as a stable deep pathway of cervical sympathetic nerves. The deep pathway (vertebral nerve and its branches) with the superficial pathways (cervical sympathetic trunk and its branches) formed a sympathetic nervous "plexus" in the cervical region. This sympathetic nervous "plexus" may be involved in the effects of cervical ganglionic blockade.

Fiber arrangements of nerves belonging to ventral and dorsal divisions in the proximal region of the brachial plexus: a study using fluorescence of DiI and DiO in adult rats.

The motor and sensory fiber arrangements in the proximal region of the spinal nerves are important for understanding the relationship of the peripheral nerves to neuron distribution. On the other hand, the fiber arrangements are also important for the treatment of peripheral nerve grafting. We studied the fiber arrangements of two divisions (ventral and dorsal) in the proximal region of the brachial plexus and the fiber arrangements of the lateral cutaneous rami in Th7 and Th8 intercostal nerves in adult rats with a method using the fluorescent pigments DiI and DiO. Results showed that fiber arrangements belonging to the two divisions have a specific separate distribution in the distal region. However, this specific separate distribution form was absent in the proximal region of the spinal nerves in the plexus. Fiber arrangements of the lateral cutaneous ramus in the anterior branches of the thoracic nerves (intercostal nerves) were also observed in comparison with those in the brachial plexus by the same method. In the intercostal nerves, fibers of the lateral cutaneous ramus were distributed in the dorsal portion from distal to proximal. These results suggest that there are two types of fiber arrangement in the proximal regions of the spinal nerves: a ventrodorsal distributional type, comprising intercostal nerves and the dorsal branches of the spinal nerves; and a mixed type, comprising the anterior branches of the cervical and brachial (and perhaps lumbar) plexuses. On the other hand, fibers of the lateral cutaneous rami in the intercostal nerves were distributed on the dorsal part of the nerves. These results of fiber arrangement analysis for the intercostal nerves may offer an opportunity to improve the effect of treatments using peripheral nerve grafting and suturing in the brachial and lumbar plexus with intercostal nerves.

Growth of collagen fibrils produced by human osteosarcoma cells: high-resolution scanning electron microscopy.

To demonstrate three-dimensionally the process of the collagen fibril growth, the bottom of culture dishes with human osteosarcoma cells (NOS-1) and their extracts were examined by conventional scanning electron microscopy (SEM). Backscattered electron (BSE) imaging of SEM was also applied to the specimens, which were stained with phosphotungustic acid and uranyl acetate. Conventional SEM images showed several stages of collagen fibril assembly. Short collagen fibrils with tapered ends were distributed at the bottom of the dish just beneath and/or around the cultured cells; they were 1 microm long and 20-30 nm in diameter at the thickest middle portion. These fibrils were often twisted and united in a right helical direction, and consequently increased in length (5-10 microm) and diameter (more than 100 nm). In BSE images, the periodical bands stained with phosphotungstic acid and uranyl acetate were visualized throughout the individual fibrils. The banding pattern indicated that the polarity of the collagen molecules was unidirectional; namely, that all molecules were pointed in the same direction throughout the length of the fibrils.

Identification of two amino acids contributing the high enzyme activity in the alkaline pH range of an alkaline endoglucanase from a Bacillus sp.

An alkaline cellulase (beta-1,4-endoglucanase; NK1) from an alkalophilic Bacillus sp. shows great similarity in amino acid sequence to a neutral cellulase (BSC) from Bacillus subtilis, despite a considerable difference in their pH activity profiles. Multiple amino acid exchanges by site-directed mutagenesis, using BSC as the reference, were performed on the residues in region 5 of NK1, which was previously shown to be responsible for the high enzyme activity of this alkaline cellulase in a broad alkaline pH range. Two amino acid residues, Ser287 and Ala296, were identified as being responsible for the activity in the alkaline range. The double mutation, Ser287 to Asn and Ala296 to Ser, of NK1 made its pH activity profile almost the same as that of BSC. On the other hand, the pH activity profile in the acidic range was not significantly affected by various amino acid replacements including these two positions in region 5. This observation, together with the information available on other endoglucanases, suggests that the above two amino acid substitutions caused a profound effect through rearrangement of the hydrogen bond network forming the substrate-binding site or the catalytic site.