Human RBP4 ELISA Matched Antibody Pair

Background: O-G1cNAcylation is an important mechanism of diabetic complication. Retinoid homeostasis regulates cell-physiological functions through STRA6-retinol signaling. Therefore, we investigated whether O-G1cNAcylation disrupted STRA6-retinol signals in diabetes. Methods: Immunoprecipitation and proximity ligation assay were used to investigate O-G1cNAcylation of STRA6-retinol signals in kidneys of db/db and ob/ob mice. Western blot and immunohistochemistry were done for STRA6/CRBP1/LRAT/RALDH1/RARs pathway, GFAT, OGT, TGF beta(1) and collagen 1 level. HPLC and ELISA for retinol, retinal, and retinoic acid concentrations were performed in vivo and vitro. RBP4 binding with STRA6 was measured in vitro. To verify whether O-G1cNAcylation disrupted STRA6-retinol signals, treatment of TMG and OSMI-1, transfection of OGA and OGT, and OGT siRNA were performed in HK-2 cells. Results: STRA6 and RALDH1 were highly O-G1cNAc-modified in glomeruli and tubules of db/db and ob/ob mice. RBP4, p-Try, p-JAK2, and p-STATS on STRA6 immunoprecipitate were reduced. Cellular retinol signals (CRBP1, LRAT, ADH, retinol, retinal, RA, RAR alpha, RAR gamma and RXR alpha) remarkably decreased in kidneys of db/db, ob/ob mice and HG-cultured cells. TMG and OGT transfection induced O-G1cNAcylation of STRA6 and RALDH1, repressed RBP4-bound STRA6, and retinol signals in NG-cultured cells. OSMI-1, OGA transfection, and OGT silence reversed O-G1cNAc-modification of STRA6 and RALDH1, and rescued the decrease of retinol signals, and reversed the increase of TGF beta(1) and collagen 1 in HG-treated cells. Conclusions: O-G1cNAcylation significantly modified STRA6 and RALDH1, suppressed RBP4 binding activity, and disrupted retinol signals in the kidney of diabetes. General significance: This study first indicates that STRA6-retinol signals were directly disrupted by O-G1cNAcylation in diabetic kidney.

Synaptosomal associated protein 25 kDa (SNAP25) is an essential component of the SNARE complex regulating synaptic vesicle fusion. SNAP25 deficiency has been implicated in a variety of cognitive disorders. We ablated SNAP25 from selected neuronal populations by generating a transgenic mouse (B6-Snap25tm3mcw (Snap25-flox)) with LoxP sites flanking exon5a/5b. In the presence of Cre-recombinase, Snap25-flox is recombined to a truncated transcript. Evoked synaptic vesicle release is severely reduced in Snap25 conditional knockout (cKO) neurons as shown by live cell imaging of synaptic vesicle fusion and whole cell patch clamp recordings in cultured hippocampal neurons. We studied Snap25 cKO in subsets of cortical projection neurons in vivo (L5-Rbp4-Cre; L6-Ntsr1-Cre; L6b-Drd1a-Cre). cKO neurons develop normal axonal projections, but axons are not maintained appropriately, showing signs of swelling, fragmentation and eventually complete absence. Onset and progression of degeneration are dependent on the neuron type, with L5 cells showing the earliest and most severe axonal loss. Ultrastructural examination revealed that cKO neurites contain autophagosome/lysosome-like structures. Markers of inflammation such as Iba1 and lipofuscin are increased only in adult cKO cortex. Snap25 cKO can provide a model to study genetic interactions with environmental influences in several disorders.