We studied bovine subjects that exhibited a moderate uncompensated anemia with hereditary spherocytosis inherited in an autosomal incompletely dominant mode and retarded growth. Based on the results of SDS-PAGE, immunoblotting, and electron microscopic analysis by the freeze fracture method, we show here that the proband red cells lacked the band 3 protein completely. Sequence analysis of the proband band 3 cDNA and genomic DNA showed a C → T substitution resulting in a nonsense mutation (CGA → TGA; Arg → Stop) at the position corresponding to codon 646 in human red cell band 3 cDNA. The proband red cells were deficient in spectrin, ankyrin, actin, and protein 4.2, resulting in a distorted and disrupted membrane skeletal network with decreased density. Therefore, the proband red cell membranes were extremely unstable and showed the loss of surface area in several distinct ways such as invagination, vesiculation, and extrusion of microvesicles, leading to the formation of spherocytes. Total deficiency of band 3 also resulted in defective Cl Ϫ /HCO 3 Ϫ exchange, causing mild acidosis with decreases in the HCO 3

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Various mutations in the AE1 (anion exchanger 1, band 3) gene cause dominant hereditary spherocytosis, a common congenital hemolytic anemia associated with deficiencies of AE1 of different degrees and loss of mutant protein from red blood cell membranes. To determine the mechanisms underlying decreases in AE1 protein levels, we employed K562 and HEK293 cell lines and Xenopus oocytes together with bovine wild-type AE1 and an R664X nonsense mutant responsible for dominant hereditary spherocytosis to analyze protein expression, turnover, and intracellular localization. R664X-mutant protein underwent rapid degradation and caused specifically increased turnover and impaired trafficking to the plasma membrane of the wild-type protein through hetero-oligomer formation in K562 cells. Consistent with those observations, co-expression of mutant and wild-type AE1 reduced anion transport by the wild-type protein in oocytes. Transfection studies in K562 and HEK293 cells revealed that the major pathway mediating degradation of both R664X and wild-type AE1 employed endoplasmic reticulum (ER)-associated degradation through the proteasomal pathway. Proteasomal degradation of R664X protein appeared to be independent of both ubiquitylation and N-glycosylation, and aggresome formation was not observed following proteasome inhibition. These findings indicate that AE1 R664X protein, which is associated with dominant hereditary spherocytosis, has a dominant-negative effect on the expression of wild-type AE1

We have recently characterized IQI/Jic mice as a model for Sjö gren's syndrome (SS), a chronic autoimmune disease in humans. In SS, local lymphocytic infiltrations into salivary and lacrimal glands frequently develop to the involvement of systemic exocrine and nonexocrine organs, and the mechanism for progression of this disease remains obscure. Herein, we report identification of an autoantigen shared by various target organs in IQI/Jic mice. Polypeptides identified based on immunorecognition by autoantibodies in sera from IQI/ Jic mice affected with autoimmune disease (>12 weeks of age) were tissue kallikrein (Klk)-1 and -13 and were cross-reactive to the autoantibodies. Interestingly, Klk-13, but not Klk-1, caused a proliferative response of splenic T cells from IQI/Jic mice from the age of 4 weeks onward. In addition, remarkably enhanced expression of Klk-13 was observed in the salivary glands of the mice in accordance with the development of inflammatory lesions. These results indicate that Klk-13 acts as an autoantigen and may increase T cells responsive to organs commonly expressing Klk-13, playing a pivotal role in the etiology of progression of disease in IQI/Jic mice. Our findings provide insights into the contributions of autoantigens shared by multiple organs in the progress of SS from an organ-specific to a systemic disorder.

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