Sorting of growth hormone–erythropoietin fusion proteins in rat salivary glands

Abstract

Neuroendocrine and exocrine cells secrete proteins in either a constitutive manner or via the regulated secretory pathway (RSP), but the specific sorting mechanisms involved are not fully understood. After gene transfer to rat salivary glands, the transgenic model proteins human growth hormone (hGH) and erythropoietin (hEpo) are secreted primarily into saliva (RSP; exocrine) and serum (constitutive; endocrine), respectively. We hypothesized that fusion of hGH at either the C-terminus or the N-terminus of hEpo would re-direct hEpo from the bloodstream into saliva. We constructed and expressed two fusion proteins, hEpo-hGH and hGH-hEpo, using serotype 5-adenoviral vectors, and delivered them to rat submandibular glands in vivo via retroductal cannulation. Both the hEpohGH and hGH-hEpo fusion proteins, but not hEpo alone, were secreted primarily into saliva (p<0.0001 and P=0.0083, respectively). These in vivo studies demonstrate for the first time that hGH, in an N-as well as C-terminal position, influences the secretion of a constitutive pathway protein.

Expression of plasmid DNA in the salivary gland epithelium: novel approaches to study dynamic cellular processes in live animals

“…Because they are easily accessible through the intraoral cannulation of the major excretory (Wharton's) duct, the SMGs are a useful target for transgene expression. Indeed, successful gene transfer into rat and mouse SMGs has been shown using viral-based (1,2,4,6,24,33,41) and non-viral-based (11,18,29,30) approaches. Viral vectors show diverse tropism for acinar and ductal cells in mouse SMGs.…”

Toward gene therapy for growth hormone deficiency via salivary gland expression of growth hormone

“…We also focused on the N‐terminus of hGH based on the original findings Moore and Kelly () using a hGH‐VSVG fusion protein and on the reported results with an hGH‐erythropoietin fusion protein (Samuni et al , ), suggesting that the N‐terminus of hGH may encode sorting properties for the RSP. We created three N‐terminal mutants, with substitutions that changed polarity and charge (Table ), and two N‐terminus deletions.…”

“…Also, point mutations and a C‐terminal extension arising from a cloning artifact demonstrated partial redirection of hGH to the CSP in SG cells in vivo (Wang et al , ). In another study (Samuni et al , ), the N‐terminal region of hGH was also shown to influence its sorting into the RSP. Our aim in this study was to use site‐directed mutagenesis in the N‐ or C‐terminus of hGH to divert its sorting from the RSP to the CSP.…”

Salivary Glands: A Powerful Experimental System to Study Cell Biology in Live Animals by Intravital Microscopy

“…Notably, for viral-mediated gene therapy in humans, the SGs offer several advantages with respect to other organs: i) the encapsulation of the SG tissue prevents the dissemination of the virus in the rest of the body (Baum et al, 2004;Voutetakis et al, 2004), ii) in case of potential health issues the SGs can be removed since they are not essential for life, iii) the differentiation of the cells provides a relatively stable cell populations for non-integrating vectors, and iv) duacrine (both exocrine and endocrine) protein secretion allows to direct the expressed molecules into either the saliva or the blood stream (Baum et al, 2004). Numerous studies have shown successful gene transfer into both rat and mouse submandibular glands using viral-based approaches, which offer the advantage of a more robust expression of the transgenes (Andresen et al, 2009;Baum and Tran, 2006;Delporte et al, 1996;Honigman et al, 2001;Mastrangeli et al, 1994;Morita et al, 2011;Palaniyandi et al, 2011;Perez et al, 2011;Samuni et al, 2008;Wang et al, 2000;Zheng et al, 2009). However, non viral-mediated approaches have also been utilized, although limited to a small percentage of the cells in the parenchyma (Goldfine et al, 1997;Honigman et al, 2001;Niedzinski et al, 2003a;Niedzinski et al, 2003b;Passineau et al, 2010;Sramkova et al, 2009).…”