Sec23b deficiency In Mice Results In Pancreatic Destruction and Defective long Term Hematopoietic Stem Cell Function
Authors: Vasievich Matthew , Zhang Bin , Rinehart Jesse , Jones Morgan , Maillard Ivan , Ginsburg David 
Published: October 2019

Abstract
Abstract 2038
Congenital Dyserythropoietic Anemia type II (CDAII) is an autosomal recessive disorder caused by mutations in the gene SEC23B. SEC23B is a component of the COPII coat complex that transports proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. CDAII is characterized by mild to moderate anemia and >10% bi- or multi- nucleate erythroblasts in the bone marrow. We generated Sec23b deficient mice (Sec23b gt/gt) from ES cells with a genetrap cassette inserted into the last intron of Sec23b. Sec23b gt/gt mice die at birth with destruction of the exocrine pancreas. Eight to 12 week old lethally irradiated C57BL/6J recipients were transplanted with 106 E17.5 fetal liver cells from either wildtype or Sec23b gt/gt mice. Mice were bled at 6, 8 and 12 weeks post transplant and no significant difference was observed in either hematocrit or hemoglobin (N=3-7 mice per group). Red blood cell (rbc) ghosts prepared from peripheral blood of transplanted mice showed no shift of Band 3 on SDS-PAGE analysis. Ghost fractions from Sec23b gt/gt recipients analyzed by western blot for the presence of residual ER proteins showed no difference between wildtype and Sec23b gt/gt fetal liver cell recipients. No significant difference was found in bi-nucleate erythroblasts in the bone marrow, myeloid:erythroid ratio or spleen mass. We quantitatively analyzed the proteome of mature rbc ghosts from Sec23b gt/gt recipients by Stabile Isotope Labeling of Amino acids in Cells (SILAC) proteomics using rbc ghosts from mice fed lysine labeled with 13C (SILAC chow). When SILAC-labeled rbc ghosts were compared by mass spectrometry analysis with ghosts from Sec23b gt/gt fetal liver cell recipients, no significant differences in abundance of rbc membrane proteins were observed. To more stringently test the ability of Sec23b gt/gt fetal liver cells to engraft, 5×105 wildtype or Sec23b gt/gt fetal liver cells were co-transplanted with 5×105 wildtype fetal liver cells expressing a GFP transgene. FACS analysis of peripheral blood obtained at 6 and 8 weeks post transplant showed significant out-competition of Sec23b gt/gt cells by GFP+ progenitors in Ter119+ rbcs (p<0.01, at all time points), CD3+ T cells (p<0.01 at 8 weeks), B220+ B cells (p<0.01, at all time points) and Mac1+Gr1+ myeloid cells (p<0.01, at all time points) compared to wildtype controls (N=5-7 mice per group). Fetal livers from wildtype and Sec23b gt/gt mice showed no difference in the total number of CD150+CD48-Sca1+cKit+(lineage-) long term hematopoietic stem cells (LT-HSCs) per fetal liver (p=0.45). In conclusion, Sec23b deficient humans and mice exhibit disparate phenotypes, apparently restricted to CDAII in humans and a prominent neonatal pancreatic insufficiency in mice. These differences could be due to evolutionary changes in relative expression and/or function of the Sec23a and b paralogs in humans and mice. However, we cannot exclude an allele-specific defect due to residual or aberrant function of the Sec23b gt allele. Analyses of a deleted and a conditional floxed Sec23b null allele are currently in progress. Finally our transplant data suggest that Sec23b traffics cargoes important for the function of murine LT-HSCs. This finding contrasts with the apparently erythroid-specific human phenotype, raising the possibility of a requirement for Sec23b function in the marrow microenvironment not tested by these transplant studies. Tissue specific deletion of Sec23b is currently in progress to address this question.

Disclosures:
No relevant conflicts of interest to declare.