Significance of IgG for the activity of factor XII measured in human plasma

The plasma level of factor XII (FXII) was measured in samples from healthy young men. The activated contact factor was assayed as prekallikrein activator (PKA), as S-2222 amidase, and in radial immunodiffusion tests. By removing the bulk of IgG on protein G columns before the activation procedure, the functional activities increased to about 135%. In such test preparations, PAGE immunoblot experiments with polyclonal antibodies against FXII showed, in addition to FXIIa (80 kD), a double band with a molecular weight of about 46 kD. This protein could also be detected with a light-chain-specific monoclonal antibody to FXII, but not with such an antibody directed against its heavy chain. The 46-kD band was also observed in plasma deficient in FXII. The amidase assays indicated that the minor part of FXIIa was present in some kind of association with another protease. To obtain a correct estimation of total FXIIa in the amidase assays a sufficiently high level of FXI was required compared to that of FXII. The PKA assays were generally carried out with a prekallikrein (PK) substrate containing IgG. By replacing this substrate by PK free from IgG additional PKA activity was observed, the activity appearing also in plasma deficient in FXII.     

Human monocyte-derived macrophages express an approximately 120-kD Ox-LDL binding protein with strong identity to CD68

A protein that specifically binds oxidized LDL (Ox-LDL) has recently been characterized in mouse peritoneal macrophages and identified as macrosialin, a protein with a molecular weight of 95 kD. First, the present work shows that human monocyte-derived macrophages express a membrane protein with a molecular weight of approximately 120 kD that selectively binds Ox-LDL. Second, we tested whether this approximately 120-kD Ox-LDL binding protein had any relation to CD68, the human homologue of macrosialin. The following evidence was obtained to support the role of CD68 as an Ox-LDL binding protein: (1) Ligand blots with Ox-LDL and Western blots with Ki-M6, an anti-human CD68 monoclonal antibody, revealed a single band with a molecular weight of approximately 120 kD under reducing and nonreducing condition. (2) The expression patterns of the approximately 120-kD Ox-LDL binding membrane protein and of CD68 paralleled each other during monocyte/macrophage differentiation. (3) Digestion with N-glycosidase F demonstrated that both CD68 and the Ox-LDL binding protein are glycoproteins; both showed a similar shift of approximately 18 kD in apparent molecular weight. (4) CD68, probed with monoclonal antibody Ki-M6, and the approximately 120-kD Ox-LDL binding protein were coprecipitated with EMB11, another anti-CD68 antibody. About 5000 molecules of CD68 are expressed on the cell surface of human macrophages. Ligation of 125I-Ki-M6 to cells leads to its internalization and degradation. This capacity would be sufficient to allow for the specific uptake and degradation of Ox-LDL. Taken together, these data support a role for CD68 as a specific Ox-LDL binding protein in human monocyte-derived macrophages.     

The 30-kD domain of protein 4.1 mediates its binding to the carboxyl terminus of pICln, a protein involved in cellular volume regulation

Erythrocyte protein 4.1 (P4.1) is an 80-kD cytoskeletal protein that is important for the maintenance of the structural integrity and flexibility of the red blood cell membrane. Limited chymotryptic digestion of erythroid P4.1 yields 4 structural domains corresponding to the 30-, 16-, 10-, and 22/24-kD domains. Using a yeast two-hybrid system, we isolated cDNA clones encoding pICln that specifically interacts with the 30-kD domain of P4.1. In this report, we show that the carboxyl-terminus (amino acid residues 103-237) of pICln binds to the 30-kD domain of P4.1 in a yeast two-hybrid system. The direct association between the 30-kD domain of P4.1 and pICln was further confirmed by the following findings: (1) the S35-methione-labeled pICln specifically bound to both GST/P4.1-80 (80 kD) and GST/P4.1-30 (30 kD) fusion proteins, but not to the proteins that lack the 30-kD domain; (2) coimmunoprecipitation analysis of the cell extracts from transfected SiHa cells showed that pICln and P4.1 associate in transfected cells. It was reported that pICln can form a complex with actin and may play a role involved in cellular volume regulation. The direct association between P4.1 and pICln suggests that pICln may link P4.1-bound cytoskeletal elements to an unidentified volume-sensitive chloride channel.     

Long-term regulation of renal urea transporter protein expression in rat

To test the hypothesis that the abundance of the apical urea transporter of the inner medullary collecting duct (IMCD) is regulated in vivo by factors associated with altered water balance, immunoblots of rat inner medullary membrane fractions were probed with rabbit polyclonal antibodies against the renal urea transporter (RUT) gene product. In inner medullas of Brattleboro rats, which manifest severe chronic water diuresis, a 117-kD band was seen, in addition to the previously described 97-kD band. These two bands were detectable by antibodies directed against two different regions of the RUT sequence. When Brattleboro rats were treated with a 5-d infusion of arginine vasopressin (AVP) by osmotic minipump, the 117-kD band was markedly diminished, whereas the 97-kD band was unchanged. Simultaneous infusion of the diuretic agent furosemide prevented the AVP-induced decrease in the 117-kD band. In AVP-infused Sprague Dawley rats, the 117-kD band was barely perceptible. However, when AVP-treated rats were infused with furosemide for 5 d, the 117-kD band was markedly accentuated, whereas the 97-kD band was unchanged. The abundance of the 117-kD RUT protein in the renal papilla was inversely correlated with dietary protein intake. Further immunoblotting studies revealed that the 117-kD protein is heavily expressed in IMCD cells and not in non-collecting duct components of the inner medulla, and is present in low-density microsome fractions from inner medulla. From this study, the following conclusions can be made: (1) The collecting duct urea transporter is present in at least two forms (97 and 117 kD) in the IMCD. (2) The expression level of the 117-kD urea transporter protein is regulated and is inversely correlated with medullary osmolality and urea concentration, but does not correlate with circulating AVP level. (3) Although AVP regulates RUT function on a short-term basis, long-term changes in AVP levels do not increase RUT abundance.     

Analysis of Huntingtin-associated protein 1 in mouse brain and immortalized striatal neurons

Huntingtin, the protein product of the Huntington's disease (HD) gene, is expressed with an expanded polyglutamine domain in the brain and in nonneuronal tissues in patients with HD. Huntingtin-associated protein 1 (HAP-1), a brain-enriched protein, interacts preferentially with mutant huntingtin and thus may be important in HD pathogenesis. The function of HAP-1 is unknown, but recent evidence supports a role in microtubule-dependent organelle transport. We examined the subcellular localization of HAP-1 with an antibody made against the NH2-terminus of the protein. In immunoblot assays of mouse brain and immortalized striatal neurons, HAP-1 subtypes A and B migrated together at about 68 kD and separately at 95 kD and 110 kD, respectively. In dividing clonal striatal cells, HAP-1 localized to the mitotic spindle apparatus, especially at spindle poles and on vesicles and microtubules of the spindle body. Postmitotic striatal neurons had punctate HAP-1 labeling throughout the cytoplasm. Western blot analysis of protein extracts obtained after subcellular fractionation and differential centrifugation of the clonal striatal cells showed that HAP-1B was preferentially enriched in membrane fractions. Electron microscopic study of adult mouse basal forebrain and striatum showed HAP-1 localized to membrane-bound organelles including large endosomes, tubulovesicular structures, and budding vesicles in neurons. HAP-1 was also strongly associated with an unusual large "dense" organelle. Microtubules were labeled in dendrites and axonal fibers. Results support a role for HAP-1 in vesicle trafficking and organelle movement in mitotic cells and differentiated neurons and implicate HAP-1B as the predominant molecular subtype associated with vesicle membranes in striatal neurons.     

Functional and immunocytochemical identification of glutamate autoreceptors of an NMDA type in crayfish neuromuscular junction

Functional and immunocytochemical identification of glutamate autoreceptors of an NMDA type in crayfish neuromuscular junction. J. Neurophysiol. 80: 2893-2899, 1998. N-Methyl--aspartate (NMDA) reduces release from crayfish excitatory nerve terminals. We show here that polyclonal and monoclonal antibodies raised against the mammalian postsynaptic NMDA receptor subunit 1 stain specifically the presynaptic membrane of release boutons of the crayfish neuromuscular junction. In crayfish ganglionic membranes, the polyclonal antibody recognizes a single protein band that is somewhat larger (by approximately 30 kD) than the molecular weight of the rat receptor. Moreover, the monoclonal (but not the polyclonal) antibody abolishes the physiological effect of NMDA on glutamate release. The monoclonal antibody did not prevent the presynaptic effects of glutamate, which also reduces release by activation of quisqualate presynaptic receptors. Only when 6-cyano-7-nitroquinoxatine-2,3,dione (CNQX) was added together with the monoclonal antibody was the presynaptic effect of glutamate blocked. These results show that presynaptic glutamate receptors of the crayfish NMDA type are involved in the regulation of neurotransmitter release in crayfish axon terminals. Although the crayfish receptor differs in its properties from the mammalian NMDA receptor, the two receptors retained some structural similarity.     

Structure, properties, and tissue localization of apoplastic alpha-glucosidase in crucifers

Apoplastic alpha-glucosidases occur widely in plants but their function is unknown because appropriate substrates in the apoplast have not been identified. Arabidopsis contains at least three alpha-glucosidase genes; Aglu-1 and Aglu-3 are sequenced and Aglu-2 is known from six expressed sequence tags. Antibodies raised to a portion of Aglu-1 expressed in Escherichia coli recognize two proteins of 96 and 81 kD, respectively, in vegetative tissues of Arabidopsis, broccoli (Brassica oleracea L.), and mustard (Brassica napus L.). The acidic alpha-glucosidase activity from broccoli flower buds was purified using concanavalin A and ion-exchange chromatography. Two active fractions were resolved and both contained a 96-kD immunoreactive polypeptide. The N-terminal sequence from the 96-kD broccoli alpha-glucosidase indicated that it corresponds to the Arabidopsis Aglu-2 gene and that approximately 15 kD of the predicted N terminus was cleaved. The 81-kD protein was more abundant than the 96-kD protein, but it was not active with 4-methylumbelliferyl-alpha-D-glucopyranoside as the substrate and it did not bind to concanavalin A. In situ activity staining using 5-bromo-4-chloro-3-indolyl-alpha-D-glucopyranoside revealed that the acidic alpha-glucosidase activity is predominantly located in the outer cortex of broccoli stems and in vascular tissue, especially in leaf traces.     

Immunologic recognition of a 25-amino acid repeat arrayed in tandem on a major antigen of Blastomyces dermatitidis

A 120-kD glycoprotein antigen abundantly expressed on Blastomyces dermatitidis yeasts is a target of cellular and humoral immune responses in human infection. To investigate the antigen and immune response more carefully at the molecular level, we screened an expression library from B. dermatitidis to identify clones that encode this antigen, designated WI-1. A 942-bp cDNA was isolated by immunologic screening with polyclonal, rabbit anti-WI-1 antiserum. Northern hybridization analysis showed that the cDNA hybridized to yeast message approximately equal to 3.9 kb. DNA and deduced protein sequence analysis of the clone demonstrated a 25-amino acid repeat arrayed in tandem, present in 4.5 copies near the 5' end, and rich in predicted antigenic epitopes. Further analysis showed strong homology in these tandem repeats with invasin, an adhesin of Yersiniae. Cloned cDNA was used to express a 30-kD fusion protein strongly recognized in western blots by rabbit anti-WI-1 antiserum, and by sera from all 35 blastomycosis patients studied. The fusion protein product of subcloned cDNA encoding only the tandem repeat also was strongly recognized in western blots by sera from the 35 blastomycosis patients, but not by sera from 10 histoplasmosis and 5 coccidioidomycosis patients. An antigen-inhibition radioimmunoassay showed that the tandem repeat alone completely eliminated rabbit and human anti-WI-1 antibody binding to radiolabeled native WI-1. From these results, we conclude that the 25-amino acid repeat of WI-1 displays an immunodominant B cell epitope, and that the carboxyl-terminus of the molecule exhibits an architecture that may promote adhesion of Blastomyces yeasts to host cells or extracellular matrix proteins and ultimately provide a clearer picture of the molecular pathogenesis of blastomycosis.     

Chicken acidic leucine-rich EGF-like domain containing brain protein (CALEB), a neural member of the EGF family of differentiation factors, is implicated in neurite formation

Chicken acidic leucine-rich EGF-like domain containing brain protein (CALEB) was identified by combining binding assays with immunological screens in the chicken nervous system as a novel member of the EGF family of differentiation factors. cDNA cloning indicates that CALEB is a multidomain protein that consists of an NH2-terminal glycosylation region, a leucine-proline-rich segment, an acidic box, a single EGF-like domain, a transmembrane, and a short cytoplasmic stretch. In the developing nervous system, CALEB is associated with glial and neuronal surfaces. CALEB is composed of a 140/130-kD doublet, an 80-kD band, and a chondroitinsulfate-containing 200-kD component. The latter two components are expressed in the embryonic nervous system and are downregulated in the adult nervous system. CALEB binds to the extracellular matrix glycoproteins tenascin-C and -R. In vitro antibody perturbation experiments reveal a participation of CALEB in neurite formation in a permissive environment.     

Brain-specific receptor-type protein-tyrosine phosphatase RPTP beta is a chondroitin sulfate proteoglycan in vivo

By using polyclonal antiserum, which recognizes multiple proteoglycan core proteins, we isolated a cDNA species for an unknown chondroitin sulfate proteoglycan in bovine brain. Unexpectedly, DNA sequencing revealed that the cDNA encodes an open reading frame highly homologous to the human receptor-type protein-tyrosine phosphatase, RPTP beta. To prove that RPTP beta is a proteoglycan, we raised three polyclonal antibodies against extracellular and cytoplasmic domains of human RPTP beta. These antibodies have been shown to react with a smear band ranging from 350 to 500 kDa in human brain extracts. Digestion with chondroitinase ABC eliminated this smear and gave rise to a 310/300-kDa doublet band that was not detected without digestion, indicating that almost all of the RPTP beta molecules in the brain contain chondroitin sulfate chains. In the cerebellum, immunofluorescence staining of chondroitinase-treated sections revealed pericellular localization of RPTP beta in the external and internal granular layers. These data establish that RPTP beta is expressed constitutively as a chondroitin sulfate proteoglycan in the brain, and suggest that chondroitin sulfates may be an essential component for the physiological function of RPTP beta in vivo.     

Phosphatidylinositol 3-kinase is necessary and sufficient for insulin-stimulated stress fiber breakdown

Rat-1 fibroblasts overexpressing the human insulin receptor undergo rapid actin rearrangement in response to insulin. Breakdown of stress fibers present in quiescent cells is followed by transient membrane ruffling and a return of stress fibers. We investigated the signaling pathways that mediate this insulin-stimulated reorganization of the actin cytoskeleton, which was visualized with rhodamine-phalloidin. Treatment of cells with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin prevented insulin action at the preliminary step of stress fiber breakdown. Cellular microinjection of a polyclonal antibody directed against the p85 subunit of PI3-kinase as well as a purified recombinant p85-SH2 domain protein also inhibited actin reorganization. Transient expression of a constitutively active form of PI3-kinase (p110*) was sufficient to cause both stress fiber breakdown and membrane ruffling in the absence of insulin. Microinjection of a polyclonal anti-Shc antibody or dominant negative N17-Ras protein did not affect actin dynamics, and although constitutively active V12-Ras caused modest cytoskeletal reorganization, this effect was blocked by pretreatment with wortmannin. In summary, activation of PI3-kinase is necessary and sufficient to stimulate actin rearrangement, indicating that PI3-kinase may initiate the only signaling cascade required for insulin to induce cytoskeletal restructuring.     

Protein kinase C and casein kinase 2 phosphorylate in vitro proteins of the annexin family from eggs of loach Misgurnus fossilis

A mixture of proteins of the annexin family was obtained from the cytoplasm of mature eggs of loach Misgurnus fossilis (by reprecipitation with acid phospholipids in the presence of Ca2+). This mixture comprised five proteins with molecular weights of 58, 38, 36, 35, and 31 kD. Polyclonal rabbit antibodies against the major 31-kD protein were obtained. Western blot analysis showed that the obtained antibodies exhibit a high specificity towards the 31-kD protein from eggs and other tissues of loach and zebrafish (Brachydanio rerio). The analysis of cDNA corresponding to the 31-kD protein by screening the zebrafish cDNA library confirmed that this protein belongs to the annexin family. Phosphorylation of the obtained annexins in vitro was studied. It is shown that the 58-kD protein is phosphorylated by casein kinase 2 (CK2), whereas the 38-, 36-, 35-, and 31-kD proteins are phosphorylated by protein kinase C (PKC).     

Demonstration of a new pathogenic mutation in human complex I deficiency: a 5-bp duplication in the nuclear gene encoding the 18-kD (AQDQ) subunit

We report the cDNA cloning, chromosomal localization, and a mutation in the human nuclear gene encoding the 18-kD (AQDQ) subunit of the mitochondrial respiratory chain complex I. The cDNA has an open reading frame of 175 amino acids and codes for a protein with a molecular mass of 23.2 kD. Its gene was mapped to chromosome 5. A homozygous 5-bp duplication, destroying a consensus phosphorylation site, in the 18-kD cDNA was found in a complex I-deficient patient. The patient showed normal muscle morphology and a remarkably nonspecific fatal progressive phenotype without increased lactate concentrations in body fluids. The child's parents were heterozygous for the mutation. In 19 other complex I-deficient patients, no mutations were found in the 18-kD gene.     

Immunohistochemical localization of the inositol 1,4,5-trisphosphate receptor in the human nervous system

A monoclonal antibody raised against the mouse cerebellar inositol trisphosphate receptor was used to study the immunohistochemical localization of this protein in the human central nervous system. As in the brain of rodents, strong immunoreactivity was found in dendrites, axon and cell bodies of Purkinje cells, as well as in nerve endings in the cerebellar and vestibular nuclei. Cerebellar efferent fibres were the only positive structures demonstrated in the brainstem and no immunostaining could be detected in the spinal cord or dorsal root ganglia. By contrast, numerous immunoreactive neurons were present in several telencephalic and diencephalic structures, including the brain cortex, hippocampus, basal ganglia, basal forebrain, amygdala and thalamus. Immunostaining of these brain neurons was weaker than that found in Purkinje cells and was evident in cell bodies and dendrites. Thus, the human brain contains a molecule cross-reacting with the mouse inositol trisphosphate receptor protein that is expressed in a pattern similar to that found in rodents. These findings can be of great importance for understanding the function of this protein in normal brain and its modifications in neuropathological disorders.     

Merlin, the neurofibromatosis type 2 gene product, and beta1 integrin associate in isolated and differentiating Schwann cells

Neurofibromatosis type 2, a disease characterized by the formation of multiple nervous system tumors, especially schwannomas, is caused by mutation in the gene-encoding merlin/schwannomin. The molecular mechanism by which merlin functions as a tumor suppressor is unknown, but is hypothesized to involve plasma membrane and cytoskeleton interaction. Several merlin antibodies were used to study merlin expression, localization, and protein association in primary cultures of rat sensory neurons, Schwann cells (SCs), and SCs grown with neurons (SC/N cultures) before and during differentiation into myelinating cells. Western blot analysis revealed that neurons predominantly expressed a 68-kD protein, but SCs expressed two additional 88- and 120-kD related proteins. Extensive immunological characterization demonstrated that the 88-kD protein shared three domains with the 68-kD merlin protein. Western blot analysis of soluble and insoluble culture fractions demonstrated that the majority of merlin and related proteins were soluble in isolated SCs and undifferentiated SC/N cultures, but became insoluble in myelinating SC/N cultures. Double immunofluorescence staining suggested that merlin translocated from the perinuclear cytoplasm in undifferentiated SCs to the subplasmalemma in differentiating SCs and partially colocalized with beta1 integrin. Finally, beta1 integrin antibody coimmunoprecipitated 68-kD merlin from isolated SC and undifferentiated SC/N cultures, but predominantly the 88-kD protein from differentiating SC/N cultures. Together, these results provide evidence that merlin interacts with beta1 integrin and that merlin localization changes from a cytosolic to cytoskeletal compartment during SC differentiation.     

Bacterial expression and immunological detection of human papillomavirus type 16 E7 protein

We have expressed an unfused E7 protein from human papillomavirus 16 into Escherichia coli by using a T7-RNA polymerase system. E7 mRNA was detected one hour after promoter induction. Western blot analysis using either a murine monoclonal antibody elicited against E7 or sera from cervical carcinoma patients demonstrated that recombinant E7 expressed in E. coli reacted to both of them, and a 21 kD band is observed as a positive signal. This protein provides a suitable material for further protein structure and immunological studies and offers a screening tool for identification of circulating antibodies in human sera.     

Nedd5, a mammalian septin, is a novel cytoskeletal component interacting with actin-based structures

The mouse Nedd5 gene encodes a 41.5-kD GTPase similar to the Saccharomyces and Drosophila septins essential for cytokinesis. Nedd5 accumulates near the contractile ring from anaphase through telophase, and finally condenses into the midbody. Microinjection of anti-Nedd5 antibody interferes with cytokinesis, giving rise to binucleated cells. In interphase and postmitotic cells, Nedd5 localizes to fibrous or granular structures depending on the growth state of the cell. The Nedd5-containing fibers are disrupted by microinjection of GTPgammaS and by Nedd5 mutants lacking GTP-binding activity, implying that GTP hydrolysis is required for its assembly. The Nedd5-containing fibers also appear to physically contact actin bundles and focal adhesion complexes and are disrupted by cytochalasin D, C3 exoenzyme, and serum starvation, suggesting a functional interaction with the actin-based cytoskeletal systems in interphase cells.