title: Partial redirection of transgenic human growth hormone secretion from rat salivary glands.
abstract: Regulated secretory pathway proteins, when delivered as transgenes to salivary glands, are secreted predominantly into saliva. This is not useful for those proteins whose therapeutic function is required systemically, for example, human growth hormone (hGH). One strategy to improve the efficiency of hGH secretion into the bloodstream involves manipulation of existing sorting signals. The C terminus of hGH is highly conserved and contains a domain similar to the regulated pathway sorting domain of pro-opiomelanocortin (POMC). We hypothesized that, similar to POMC, mutation of this domain would divert hGH secretion from the regulated to the constitutive pathway, which in salivary glands leads to the bloodstream. Several mutations were made in the C terminus of the hGH cDNA and tested in vitro. One biologically active mutant containing E174A and E186A substitutions, and with an included C-terminal extension, was studied in greater detail. Compared with wild-type hGH, we found that this mutant hGH accumulated in the Golgi/trans-Golgi network and showed increased basal secretion in AtT20 cells, a model endocrine cell line. Importantly, in vivo, the mutant hGH displayed a relative increase in the proportion of constitutive pathway secretion seen from rat salivary glands, with a significantly lower saliva-versus-serum secretion ratio (p=0.03). Although this mutant is unlikely to be therapeutically beneficial, these results suggest that the final destination of a transgenic secretory protein may be controlled by reengineering its sorting determinants.
authors.affiliation: Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA.
authors.forename: Jianghua
authors.initials: J
authors.lastname: Wang
authors.forename: Niamh X
authors.initials: NX
authors.lastname: Cawley
authors.forename: Antonis
authors.initials: A
authors.lastname: Voutetakis
authors.forename: Yazmin M
authors.initials: YM
authors.lastname: Rodriguez
authors.forename: Corinne M
authors.initials: CM
authors.lastname: Goldsmith
authors.forename: Lynnette K
authors.initials: LK
authors.lastname: Nieman
authors.forename: A T M Shamsul
authors.initials: AT
authors.lastname: Hoque
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Frank
authors.forename: Chris R
authors.initials: CR
authors.lastname: Snell
authors.forename: Y Peng
authors.initials: YP
authors.lastname: Loh
authors.forename: Bruce J
authors.initials: BJ
authors.lastname: Baum
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D019382:Human Growth Hormone / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000378:metabolism; D012469:Salivary Glands / Q000378:metabolism*; D014161:Transduction, Genetic; D019076:Transgenes
chemical_list: D011994:Recombinant Proteins; D019382:Human Growth Hormone; D000409:Alanine