Abstract
In a search of new, small leucine-rich repeat proteoglycan/protein (SLRP) family members, a novel gene, nephrocan (NPN), has been identified. The gene consists of three exons, and based on the deduced amino acid sequence, NPN has 17 leucine-rich repeat motifs and unique cysteine-rich clusters both in the N and C termini, indicating that this gene belongs to a new class of SLRP family. NPN mRNA was predominantly expressed in kidney in adult mice, and during mouse embryogenesis, the expression was markedly increased in 11-day-old embryos at a time when early kidney development takes place. In the adult mouse kidney, NPN protein was located in distal tubules and collecting ducts. When NPN was overexpressed in cell culture, the protein was detected in the cultured medium, and upon treatment with N-glycosidase F, the molecular mass was lowered by approximately 14 kDa, indicating that NPN is a secreted N-glycosylated protein. Furthermore, transforming growth factor-beta (TGF-beta)-responsive 3TP promoter luciferase activity was down-regulated, and TGF-beta-induced Smad3 phosphorylation was also inhibited by NPN, suggesting that NPN suppresses TGF-beta/Smad signaling. Taken together, NPN is a novel member of the SLRP family that may play important roles in kidney development and pathophysiology by functioning as an endogenous inhibitor of TGF-beta signaling.
Highlights
Leucine-rich repeat (LRR)2 is a highly conserved motif from bacteria to mammals, and LRR proteins have diverse structural, biological, and cellular functions such as matrix organization, host defense, inflammation, and cell death [1,2,3]
NPN Belongs to a New Class of the small leucine-rich repeat proteoglycan/ protein (SLRP) Family—Based on the predicted amino acid sequences, NPN is a 512-amino acidlong protein and shares some structural characteristics with other SLRP members, i.e. it has a putative signal peptide sequence, a characteristic cysteine cluster (CX3CXCX7C) in the N terminus (Fig. 1A), 17 LRR motifs (Fig. 1B), and 3 exons (Fig. 1C)
In NPN, of 17 LRR motifs, there are 11 type T and 6 type S present (Fig. 1B). Based on these criteria [27], class I and II SLRPs have 12 LRR motifs composed of four tandem STT “super-motifs,”, i.e. (STT)4
Summary
Cell Culture—Human embryonic kidney 293 cells were purchased from Clontech and maintained in Dulbecco’s modified Eagle’s medium (Invitrogen) containing a high concentration of glucose (4.5 mg/ml), supplemented with 10% fetal bovine serum (Sigma), 100 units/ml penicillin, and 100 g/ml streptomycin in a 5% CO2 atmosphere at 37 °C. Tissue Distribution Determined by RT-PCR and Real Time PCR—RT-PCR was performed using the mouse MTC panel I (BD Biosciences) containing cDNA of heart, brain, spleen, lung, liver, skeletal muscle, kidney, testis, 7-, 11-, 15-, and 17-day-old embryo as templates. Generation and Characterization of Anti-NPN Antibody—A polyclonal NPN antibody was generated by immunizing rabbits with a synthetic peptide corresponding to the sequence in the 4th LRR region of NPN, i.e. 112SALPANLEVLKLNDNAIC129 (Alpha Diagnostic International Inc., San Antonio, TX) Both preimmune serum and anti-NPN polyclonal antibody were further affinity-purified with ImmunoPure (A Plus) IgG purification kit (Pierce) and used for Western blot analyses. Cultured media were collected and subjected to Western blot analyses with anti-V5 antibody to assess the NPN-V5/His protein levels synthesized by the clones.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
