Checkpoint-Independent Regulation of Origin Firing by Mrc1 through Interaction with Hsk1 Kinase.

Mrc1 is a conserved checkpoint mediator protein that transduces the replication stress signal to the downstream effector kinase. The loss of mrc1 checkpoint activity results in the aberrant activation of late/dormant origins in the presence of hydroxyurea. Mrc1 was also suggested to regulate orders of early origin firing in a checkpoint-independent manner, but its mechanism was unknown. Here we identify HBS (Hsk1 bypass segment) on Mrc1. An ΔHBS mutant does not activate late/dormant origin firing in the presence of hydroxyurea but causes the precocious and enhanced activation of weak early-firing origins during normal S-phase progression and bypasses the requirement for Hsk1 for growth. This may be caused by the disruption of intramolecular binding between HBS and NTHBS (N-terminal target of HBS). Hsk1 binds to Mrc1 through HBS and phosphorylates a segment adjacent to NTHBS, disrupting the intramolecular interaction. We propose that Mrc1 exerts a "brake" on initiation (through intramolecular interactions) and that this brake can be released (upon the loss of intramolecular interactions) by either the Hsk1-mediated phosphorylation of Mrc1 or the deletion of HBS (or a phosphomimic mutation of putative Hsk1 target serine/threonine), which can bypass the function of Hsk1 for growth. The brake mechanism may explain the checkpoint-independent regulation of early origin firing in fission yeast.

Epitope analysis of Japanese cedar pollen allergen Cry j2 with a human IgM class monoclonal antibody 404-117.

Japanese cedar pollen allergen Cry j2 is a causal allergen of seasonal pollinosis in Japan. To analyze B cell epitopes of Cry j2, we established two human-mouse hybridomas secreting IgM class human monoclonal antibodies to Cry j2. A pin-peptide enzyme-linked immunosorbent assay with synthesized icosa peptides showed that 404-117 monoclonal antibody bound to peptides #11-13 with cry j2 amino acid sequence of 101F-L140. Detailed analysis with octa peptides and alanine substituted peptides indicated that an amino acid sequence of 118FKVD121 was an essential for antibody binding. When K119 (Asn) was substituted with alanine, 404-117 monoclonal antibody did not bind to the alanine substituted peptide. We concluded that the 118FKVD121 sequence might have a very important role in early recognition by Cry j2-specific B cells, which could act as antigen presenting cells.

Epitope analysis of Japanese cedar pollen allergen Cry j1 with the human monoclonal antibody 4701-1.

We obtained a stable human-mouse hybridoma clone 4701-1 secreting IgM class human monoclonal antibody to Japanese cedar pollen allergen Cry j1. A pin-peptide enzyme-linked immunosorbent assay (ELISA) with synthesized pentadeca peptides showed a peptide with an amino acid sequence of LYTVT NSDDD PVNPA was found to be positive. Detailed analysis with deca to tetra peptides indicated that an amino acid sequence of TVTN was an essential sequence for antibody binding. When N (Asn) was substituted with A (Ala) of the TVTN epitope, the resulting peptide did not have antibody binding ability. We concluded that the TVTN sequence might have a very important role in early recognition of Cry j1 allergen by Cry j1-specific B cells, which act as antigen presenting cells.

Hsk1 kinase and Cdc45 regulate replication stress-induced checkpoint responses in fission yeast.

In fission yeast, replication fork arrest activates the replication checkpoint effector kinase Cds1(Chk2/Rad53) through the Rad3(ATR/Mec1)-Mrc1(Claspin) pathway. Hsk1, the Cdc7 homologue of fission yeast required for efficient initiation of DNA replication, is also required for Cds1 activation. Hsk1 kinase activity is required for induction and maintenance of Mrc1 hyperphosphorylation, which is induced by replication fork block and mediated by Rad3. Rad3 kinase activity does not change in an hsk1 temperature-sensitive mutant, and Hsk1 kinase activity is not affected by rad3 mutation. Hsk1 kinase vigorously phosphorylates Mrc1 in vitro, predominantly at non-SQ/TQ sites, but this phosphorylation does not seem to affect the Rad3 action on Mrc1. Interestingly, the replication stress-induced activation of Cds1 and hyperphosphorylation of Mrc1 is almost completely abrogated in an initiation-defective mutant of cdc45, but not in an mcm2 or polε mutant. The results suggest that Hsk1-mediated loading of Cdc45 onto replication origins may play important roles in replication stress-induced checkpoint.

Interactions between Swi1-Swi3, Mrc1 and S phase kinase, Hsk1 may regulate cellular responses to stalled replication forks in fission yeast.

The Swi1-Swi3 replication fork protection complex and Mrc1 protein are required for stabilization of stalled replication forks in fission yeast. Hsk1 kinase also plays roles in checkpoint responses elicited by arrested replication forks. We show that both Swi1 and Swi3, the abundance of which are interdependent, are required for chromatin association of Mrc1. Co-immunoprecipitation experiments show the interactions of Swi1-Swi3, Mrc1 and Hsk1. Mrc1 interacts with Swi3 and Hsk1 proteins through its central segment (378-879) containing a SQ/TQ cluster, and this segment is sufficient for checkpoint reaction. The SQ/TQ cluster segment (536-673) is essential but not sufficient for the interactions and for resistance to replication inhibitor hydroxyurea. Mrc1 protein level is increased in hsk1-89 cells due to apparent stabilization, and we have identified a potential phosphodegron sequence. These results suggest that interactions of the Swi1-Swi3 complex and Hsk1 kinase with Mrc1 may play a role in cellular responses to stalled replication forks in fission yeast.

Epitope analysis of peanut allergen Ara h1 with oligoclonal IgM antibody from human B-lymphoblastoid cells.

To analyze epitopes of peanut allergen Ara h1, Epstein-Barr virus-transformed human peripheral oligoclonal B-cells were cultured to obtain antibodies to Ara h1. The combined reaction pattern with six oligoclonal antibodies showed there were six antibody binding areas named a to f in Ara h1. We found the novel antibody binding area named "area c" (171-230aa).

Generation of mouse-human hybridomas secreting human monoclonal antibodies to Japanese cedar pollen allergen Cry j1.

Peripheral blood lymphocytes from 13 donors were transformed with Epstein-Barr virus (EBV) to establish immortalized human B-cells secreting antibodies to Japanese cedar (Cryptomeria japonica) pollen allergens. EBV-transformed B-lymphocytes were then fused with mouse myeloma SP2/O3, and three clones of mouse-human hybridomas secreting human IgM class monoclonal antibodies to a cedar pollen allergen Cry j1 were established.

Expression of lysosomal protective protein/cathepsin A in a stably transformed human neuroblastoma cell line during bi-directional differentiation into neuronal and Schwannian cells.

Human neuroblastoma GOTO cell lines were established that stably express recombinant human lysosomal protective protein/cathepsin A (PPCA) cDNA by transfection. Intracellular cathepsin A (acid serine carboxypeptidase) activity increased four-fold compared with in those of the parent and mock-transfected cell lines. The immunoreactive 54 kDa precursor/zymogen and mature 32/20 kDa two-chain forms were produced in the cells. The amount of the latter form expressed in the GOTO cells was significantly larger than those in the PPCA-overexpressing CHO cell lines previously established. The intracellular proteins showed a typical lysosomal granular distribution and the glycosylated 54 kDa precursor was secreted into the culture medium without the addition of an alkalizing agent. The PPCA-overexpressing cell lines also retained the ability to differentiate bi-directionally as well as the parent cells; into neuronal cells on induction by dibutyryl cAMP in serum-free medium and into Schwannian cells on induction by bromodeoxyuridine. During the course of differentiation into neuronal and Schwannian cells, the intracellular cathepsin A activity further increased two and five times, respectively, which was associated with an increase in the expression of the 32/20 kDa two-chain form. The glycosylated precursor proteins were taken up via the mannose 6-phosphate receptors, and the cathepsin A, alpha-neuraminidase and beta-galactosidase (beta-Gal) activities deficient in the fibroblasts derived from a patient with PPCA deficiency (galactosialidosis) were restored. These results suggest that the bi-directional differentiation of GOTO cell lines stably expressing the recombinant human PPCA gene could be a model system for analyzing the functions of PPCA in peripheral neuronal cells and Schwannian cells as well as the recombinant PPCA could be a useful source for enzyme replacement therapy (ERT) for galactosialidosis patients.

Generation of mouse-human hybridomas secreting antibodies against peanut allergen Ara h1.

Two clones of mouse-human hybridomas, secreting human monoclonal antibodies to a peanut allergen Ara h1, were generated from human peripheral blood lymphocytes transformed with Epstein--Barr virus, followed by cell fusion with mouse myeloma cells. Epitope analysis with overlapping peptides synthesized on a multi-pin apparatus revealed antibody-binding sequences of Ara h1 protein.