Kimita Suyama

LC3-dependent Intracellular Membrane Tubules Induced by γ-Protocadherins A3 and B2 A ROLE FOR INTRALUMINAL INTERACTIONS

Clustered protocadherins (Pcdhs) are a family of cadherin-like molecules arranged in gene clusters (alpha, beta, and gamma). gamma-Protocadherins (Pcdh-gammas) are involved in cell-cell interactions, but their prominent intracellular distribution in vivo and different knock-out phenotypes suggest that these molecules participate in still unidentified processes. We found using correlative light and electron microscopy that Pcdh-gammaA3 and -gammaB2, but not -gammaC4, -alpha1, or N-cadherin, generate intracellular juxtanuclear membrane tubules when expressed in cells. These tubules recruit the autophagy marker MAP1A/1B LC3 (LC3) but are not associated with autophagic vesicles. Lipidation of LC3 is required for its coclustering with Pcdh-gamma tubules, suggesting the involvement of an autophagic-like molecular cascade. Expression of wild-type LC3 with Pcdh-gammaA3 increased tubule length whereas expression of lipidation-defective LC3 decreased tubule length relative to Pcdh-gammaA3 expressed alone. The tubules were found to emanate from lysosomes. Deletion of the luminal/extracellular domain of Pcdh-gammaA3 preserved lysosomal targeting but eliminated tubule formation whereas cytoplasmic deletion eliminated both lysosomal targeting and tubule formation. Deletion of the membrane-proximal three cadherin repeats resulted in tubes that were narrower than those produced by full-length molecules. These results suggest that Pcdh-gammaA and -gammaB families can influence the shape of intracellular membranes by mediating intraluminal interactions within organelles.Clustered protocadherins (Pcdhs) are a family of cadherin-like molecules arranged in gene clusters (α, β, and γ). γ-Protocadherins (Pcdh-γs) are involved in cell-cell interactions, but their prominent intracellular distribution in vivo and different knock-out phenotypes suggest that these molecules participate in still unidentified processes. We found using correlative light and electron microscopy that Pcdh-γA3 and -γB2, but not -γC4, -α1, or N-cadherin, generate intracellular juxtanuclear membrane tubules when expressed in cells. These tubules recruit the autophagy marker MAP1A/1B LC3 (LC3) but are not associated with autophagic vesicles. Lipidation of LC3 is required for its coclustering with Pcdh-γ tubules, suggesting the involvement of an autophagic-like molecular cascade. Expression of wild-type LC3 with Pcdh-γA3 increased tubule length whereas expression of lipidation-defective LC3 decreased tubule length relative to Pcdh-γA3 expressed alone. The tubules were found to emanate from lysosomes. Deletion of the luminal/extracellular domain of Pcdh-γA3 preserved lysosomal targeting but eliminated tubule formation whereas cytoplasmic deletion eliminated both lysosomal targeting and tubule formation. Deletion of the membrane-proximal three cadherin repeats resulted in tubes that were narrower than those produced by full-length molecules. These results suggest that Pcdh-γA and -γB families can influence the shape of intracellular membranes by mediating intraluminal interactions within organelles.

Use of RhD fusion protein expressed on K562 cell surface in the study of molecular basis for D antigenic epitopes

The human D antigens, one of the most clinically important blood groups, are presented by RhD protein with a putative 12 transmembrane topology. To understand the molecular basis for the complex antigenic profile of RhD protein, we expressed a series of RhD fusion proteins using different portions of Duffy protein as a tag in erythroleukemic K562 cells. Because the reactivity of monoclonal anti-RhD antibody, LOR15C9, depends mainly on the sequence coded by exon 7 of RhD, we altered DNA sequence corresponding to the amino acid residues 323–331(A) and 350–354(B) in the exon 7. The mutation in region B resulted in a severe reduction in LOR15C9 binding by flow cytometry analysis, suggesting that region B may play an important role in constituting antigen epitopes recognized by LOR15C9. On the other hand, a slight decrease in the antibody binding was observed for the region A mutant, suggesting that the intracellularly located region A may elicit a long distance effect on the formation of exofacial antigen epitopes. In addition, using various monoclonal antibodies against RhD, we compared the antigenic profile of expressed RhD fusion protein with that of endogenous RhD in K562 cells as well as in erythrocytes.

Effects of Murine Tumor Necrosis Factor on Friend Erythroleukemic Cells

Tumor necrosis inducing factor (TNF), a 140,000 molecular weight glycoprotein present in the serum of Corynebacterium parvum endotoxin-treated mice, was cytotoxic toward Friend virus-transformed erythroleukemic cells (FELC). These cells grow in culture as undifferentiated pro-erythroblasts but can be induced to differentiate in a limited fashion along the erythroid pathway to orthochromatic normoblasts by various agents such as dimethylsulfoxide (DMSO). Partially and highly purified preparations of TNF were cytotoxic toward logarithmically growing FELC whereas a comparable serum protein fraction from C. parvum treated mice or endotoxin from E. coli had no effect upon FELC viability. DMSO-induced cells were more sensitive to the action of TNF requiring only about half the concentration needed to produce 50% kill in noninduced cells. Inhibition of hemoglobin formation was TNF dose-related and could be decreased by 94%. TNF was also cytotoxic toward DMSO-induced cells in stationary phase and mitomycin C treated noninduced FELC. Neuraminidase modification of the surface of FELC increased the cytotoxicity of TNF by 50%. These results demonstrate that TNF destroys FELC whether they are nondividing, dividing or partially differentiated and suggest that TNF may accomplish this by affecting cell metabolism after internalization.

Expression of the Rh-Related Glycoprotein (Rh50)

The Rh50 glycoprotein is suspected of being involved in Rh antigen expression. We prepared Rh50 cDNA from a human bone marrow library by polymerase chain reaction and then subcloned this cDNA into various vectors. The vector containing Rh50 cDNA produced a 30-kDa nonglycosylated form of Rh50 in a rabbit reticulocyte lysate system and produced partially glycosylated Rh50 (32 kDa) when microsomes were added to the system. COS-1 cells transiently transfected with the vector containing Rh50 cDNA produced partially glycosylated Rh50 (32 kDa) recognized by a Rh50-specific antibody. Surface expression of Rh50 in K562 cells was also detected by flow cytometry using mouse monoclonal antibody (2D10) specific to Rh50. Partially glycosylated Rh50 (32 kDa) was again isolated from the lysates of K562 cells metabolically labeled with [35S]-methionine or [3H]-mannose using anti-Rh50 antisera. These systems (K562 and COS-1 cells) should prove useful for studying the transport of Rh proteins within the cell and the necessary components needed for Rh antigenicity at the cell surface.

Red cell surface cysteine residue (285) of D polypeptide is not essential for D antigenicity

BACKGROUND: Only one surface cysteine residue (285) has been thought to be involved in D antigenicity, according to studies using lyophilized or nonlyophilized red cell membranes. However, it has been reported that a 17‐kDa chymotryptic fragment containing the N‐terminus but not this cysteine residue is associated with D antigenicity.

Expression of Rh30 and Rh-related glycoproteins during erythroid differentiation in a two-phase liquid culture system.

BACKGROUND: To gain insight into the formation of the Rh complex during erythroid differentiation, the ways in which Rh30 and Rh‐related glycoproteins, especially Rh50, were produced in a modified two‐phase liquid culture system were studied.

Antitumor Activity of Normal Human Globulin: Effects on Murine and Human Erythroleukemic Cells

The effect of the antitumor fraction isolated from the alpha 2-globulin region of normal human serum (NHG-I) upon murine (FELC) and human (K562) erythroleukemic cells in vitro was determined. NHG-I inhibited the growth of both actively growing FELC and K562 cells in a dose-dependent manner. However, it has no effect upon mitomycin C treated FELC which were unable to divide nor upon dimethylsulfoxide-induced FELC in stationary phase. These results indicate that NHG-I has a cytostatic effect upon cell growth and suggests that its action may be dependent upon DNA synthesis. This is in marked contrast to TNF, the alpha 2-globulin factor obtained from murine serum which is also not species-specific but whose action upon these cell lines is cytotoxic.