Takahisa Hachiya

Identification of a novel nuclear speckle-type protein, SPOP

A novel antigen recognized by serum from a scleroderma patient was identified by expression cloning from the HeLa cell cDNA library. The cloned cDNA encoded a 374‐amino acid protein with a relative molecular mass of 47 000 and a predicted amino acid sequence 62.7% identical to the hypothetical protein of Caenorhabditis elegans, T16H12.5. The deduced amino acid sequence had a typical POZ domain and an unidentified region conserved during evolution. No zinc finger or RNA recognition motifs were found in this clone. The 2 kbp mRNA encoding the novel clone SPOP (speckle‐type POZ protein) was found to be expressed in all human tissues examined. HA‐tagged SPOP, transfected and overexpressed in COS7 cells, exhibited a discrete speckled pattern in the nuclei and was co‐localized with the splicing factor, snRNP B′/B. Deletion analysis revealed that both the POZ domain and the evolutionarily conserved region at the amino‐terminus are required for the nuclear speckled accumulation of SPOP.

Pathogenic epitopes of autoantibodies in pemphigus reside in the amino-terminal adhesive region of desmogleins which are unmasked by proteolytic processing of prosequence.

Pemphigus targets desmogleins (Dsgs), which are thought to be synthesized as inactive precursor proteins with prosequences that are cleaved by substilisin-like proprotein convertases, such as furin, to yield mature adhesive molecules. We hypothesized that some pemphigus pathogenic antibodies (Abs), which presumably interfere with adhesion, only bind the mature form. A pathogenic and three non-pathogenic anti-Dsg1 monoclonal Abs (mAbs) isolated from a pemphigus foliaceus (PF) patient, were used for immunoprecipitation and ELISA of recombinant precursor and mature Dsg1. The pathogenic Ab binds mature Dsg1, whereas non-pathogenic Abs bind either only the precursor or both the precursor and mature Dsg1. Competition ELISA showed that the majority of PF sera target the same or nearby epitopes defined by the pathogenic anti-Dsg1 mAb that blocked >20% binding of 29 out of 40 PF sera. Furthermore, the immunoreactivity of 45 PF sera against the mature Dsg1 was 3.2 fold stronger than that against the precursor Dsg1 by ELISA. Similar results were observed in anti-Dsg3 Abs in 47 pemphigus vulgaris sera, suggesting that most pemphigus sera target epitopes that are unmasked by proteolytic processing. These findings support the idea that at least some pathogenic pemphigus autoantibodies induce the loss of cell adhesion by directly binding the trans-interaction site of Dsgs.

Pathogenic anti-desmoglein MAbs show variable ELISA activity because of preferential binding of mature versus proprotein isoforms of desmoglein 3.

TO THE EDITOR The desmosomal cadherins desmoglein (DSG) 3 and DSG1 are targets of autoantibodies in the potentially fatal blistering disease, pemphigus vulgaris (PV) (Stanley and Amagai, 2006). DSGs are synthesized as preproproteins, which are processed first in the endoplasmic reticulum to remove the signal sequence and subsequently by the Golgi proprotein convertases to remove the propeptide before transport to the cell surface. The cadherin propeptide is thought to modulate the conformation of the extracellular domains to prevent intracellular aggregation because of interaction with other cadherins within the secretory pathway. Propeptide cleavage occurs upstream of the conserved tryptophan residue at position 2, which is responsible for cadherin strand dimer formation, suggesting that propeptide removal may unmask residues important in intermolecular adhesion. The proprotein convertase furin processes recombinant DSGs in baculoviral overexpression systems (Posthaus et al., 2003), which are widely used for pemphigus research and clinical diagnostic purposes. Commercial DSG ELISA kits use baculovirally produced recombinant DSG antigen (Ag) and have been shown to be a sensitive and specific diagnostic tool for pemphigus (Ishii et al., 1997). Abbreviations: Ag, antigen; DSG3, desmoglein 3; PV, pemphigus vulgaris

Letter to the EditorPathogenic Anti-Desmoglein MAbs Show Variable ELISA Activity because of Preferential Binding of Mature versus Proprotein Isoforms of Desmoglein 3

TO THE EDITOR The desmosomal cadherins desmoglein (DSG) 3 and DSG1 are targets of autoantibodies in the potentially fatal blistering disease, pemphigus vulgaris (PV) (Stanley and Amagai, 2006). DSGs are synthesized as preproproteins, which are processed first in the endoplasmic reticulum to remove the signal sequence and subsequently by the Golgi proprotein convertases to remove the propeptide before transport to the cell surface. The cadherin propeptide is thought to modulate the conformation of the extracellular domains to prevent intracellular aggregation because of interaction with other cadherins within the secretory pathway. Propeptide cleavage occurs upstream of the conserved tryptophan residue at position 2, which is responsible for cadherin strand dimer formation, suggesting that propeptide removal may unmask residues important in intermolecular adhesion. The proprotein convertase furin processes recombinant DSGs in baculoviral overexpression systems (Posthaus et al., 2003), which are widely used for pemphigus research and clinical diagnostic purposes. Commercial DSG ELISA kits use baculovirally produced recombinant DSG antigen (Ag) and have been shown to be a sensitive and specific diagnostic tool for pemphigus (Ishii et al., 1997). Abbreviations: Ag, antigen; DSG3, desmoglein 3; PV, pemphigus vulgaris

Expression patterns of DNA replication enzymes and the regulatory factor DREF during Drosophila development analyzed with specific antibodies

Summary— Specific antibodies were prepared against Drosophila DNA polymerase e and DREF, a regulatory factor for DNA replication‐related genes. Using these antibodies together with those for DNA polymerase α and proliferating cell nuclear antigen (PCNA), we examined expression patterns and sub‐cellular distributions of these proteins during Drosophila development. DNA polymerase α, ε and PCNA proteins were maternally stored in unfertilized eggs and maintained at high levels during embryogenesis. With distinct nuclear localization, proteins were observed in embryos at interphase stages throughout the 13 nuclear division cycles, suggesting that they all participate in rapid nuclear DNA replication during these cycles. In contrast, maternal storage of a DREF protein was relatively low and its level increased throughout embryogenesis. Strong nuclear staining with the anti‐DREF antibody was not observed until the nuclear division cycle 8. Immunostaining of various larval tissues from transgenic flies carrying the PCNA gene promoter‐lacZ fusion gene revealed co‐expression of DREF, PCNA and lacZ, suggesting that DREF regulates the expression of PCNA gene in these tissues. In addition, we detected a relatively high level of DREF in adult males as well as females. Since DNA polymerase α, ε and PCNA are hardly detectable in adult males, DREF very likely regulates genes other than those closely linked to DNA replication in adult males.

Type-specific expression of protein kinase C isozymes in CNS tumor cells

We examined specific expression of protein kinase C (PK-C) isozymes in cultured human glial and neuronal cell lines, using type-specific monoclonal antibodies MC-1a, -2a, and -3a (Hidaka H. et al., J. Biol. Chem., 263 (1988) 4523-4526). Immunoblotting experiments revealed that a 80 kDa band of three kinds of glioblastoma cells (A-172, SK-MG-1, SK-MG-4) was stained with MC-3a, whereas that of neuroblastoma cells (SK-N-MC) reacted with MC-2a. Immunoenzymetric assay showed that glioblastoma cells (A-172, SK-MG-1, SK-MG-4) contained 127.6 +/- 14.4, 248.8 +/- and 148.5 +/- 35.8 ng/mg protein of type III. respectively, while neuroblastoma cells (SK-N-MC) contained 389.5 +/- 20.7 ng/mg protein of type II. These results suggest that PK-C isozymes may be specifically expressed, depending on types of central nervous system (CNS) tumor cells.

Predominant expression of the src homology 2-containing tyrosine phosphatase protein SHP2 in vascular smooth muscle cells

Abstractsrc homology 2 (SH2)-containing protein-tyrosine phosphatase SHP2 is known to transduce positive signals from activated receptor protein-tyrosine kinases such as platelet-derived growth factor receptor (PDGFR) β and insulin receptor. Here, we demonstrate the physiological expression of SHP2 in rats. In northern and western blot analyses, SHP2 expressions were recognized in all tissues, but their expression levels varied significantly among tissues; it is lowest in the liver and kidney. Immunohistochemical staining and in situ hybridization showed SHP2 was expressed ubiquitously but predominantly in vascular smooth muscle cells (SMC). During the development of granulations, SHP2 was expressed predominantly in vascular SMC and also highly expressed in capillary cells. The functional associations of SHP2 with PDGFRβ, which transduces major growth signals in vascular SMC, identify a crucial function of SHP2 in blood vessels in consert with PDGFRβ.

Phosphorylation of caldesmon

The phosphorylation of caldesmon was studied to determine if kinase activity reflected either an endogenous kinase or caldesmon itself. Titration of kinase activity with calmodulin yielded maximum activity at substoichiometric ratios of calmodulin/caldesmon. The sites of phosphorylation on caldesmon for calcium/calmodulin‐dependent protein kinase II and endogenous kinase were the same, but distinct from protein kinase C sites. Phosphorylation in the presence of Ca2+ and calmodulin resulted in a subsequent increase of endogenous kinase activity in the absence of Ca2+. These results suggest that caldesmon is not a protein kinase and that kinase activity in caldesmon preparations is due to calcium/calmodulindependent protein kinase II.

Identification of type III protein kinase C in bovine aortic tissue.

We identified a subtype of protein kinase C in bovine aortic tissue. In Western blots, both the soluble and the particulate fractions from the aorta reacted only with MC-3a. In the case of hydroxylapatite column chromatography, a single activity peak of protein kinase C from the soluble and the particulate fractions was obtained with about 140 mM of potassium phosphate, a finding similar to that with the Type III protein kinase C from rabbit brain. The sandwich-type enzyme immunoassay for protein kinase C, with which the contents of each protein kinase C isozyme can be determined in the crude extracts, revealed that the Type III bovine aortic protein kinase C included 25.9 ng/mg protein. These results strongly suggest that it is the Type III protein kinase C which is mainly expressed in aortic tissue. Kinetic parameters of the Type III protein kinase C of the soluble and the particulate fractions, with respect to the Km for ATP, were 33 and 15 microM and the Km values for myosin light chain from chicken gizzard were 6.3 and 4.6 microM, respectively.

Clinical and immunological profiles of anti-BP230-type bullous pemphigoid: Restriction of epitopes to the C-terminal domain of BP230, shown by novel ELISAs of BP230-domain specific recombinant proteins

ObjectivesTo confirm that sera from some BP patients reactive exclusively to the BP230 and to study the clinical and immunological characteristics of this condition.Materials and methodsBP patients were divided into three groups: BP reactive only to BP230 (BP230- BP), BP reactive to both BP180 and BP230 (BP180-BP230-BP) and BP reactive only to BP180 (BP180-BP), based on the results of standard ELISAs for BP180 and BP230. Clinical features were statistically analyzed among the three groups. Then, targeted epitopes in each group were studied by immunoblotting and novel ELISAs using three domainspecific BP230 recombinant proteins.ResultsForty-one, 65 and 47 of 153 BP patients were categorized as BP230-BP, BP180-BP230-BP and BP180-BP, respectively. Clinically, BP230-BP patients showed significantly lower severity, less need of systemic steroids and better responses to various treatments, suggesting that BP230-BP is a milder condition. Immunoblotting and ELISAs of domain-specific BP230 recombinant proteins indicated that, while BP180-BP230-BP sera reacted with all three domains of BP230, BP230-BP sera reacted more frequently with epitopes in the BP230 C-terminal domain.ConclusionWe propose a new disease entity, named anti-BP230-type BP, in which anti-BP230 antibodies might be pathogenic and react specifically with the BP230 C-terminal domain. While anti-BP230 antibodies in BP180-BP230-BP seem to be produced via intermolecular epitope spreading, anti-BP230 antibodies in BP230-BP are considered to be produced by different mechanisms.