Ablation of GalNAc-4-sulfotransferase-1 enhances reproduction by altering the carbohydrate structures of luteinizing hormone in mice

Luteinizing hormone (LH), produced in the anterior lobe of the pituitary, is a member of the hypothalamic-pituitary-gonad axis that is required for production of the sex hormones estradiol, progesterone, and testosterone. Perturbations in levels of hormones associated with this axis can result in defects in sexual development and maturity. LH bears unique N-linked carbohydrate units that terminate with a sulfated N-acetylgalactosamine structure (GalNAc-4-SO(4)) that mediates its clearance from the blood. To determine the significance of this terminal structure, we ablated the gene encoding the sulfotransferase responsible for sulfate addition to GalNAc on LH, GalNAc-4-sulfotransferase-1 (GalNAc-4-ST1) in mice. Mice lacking GalNAc-4-ST1 exhibited increased levels of circulating LH. In male mice, this resulted in elevated levels of testosterone and precocious maturation of testis and seminal vesicles. Female mice lacking GalNAc-4-ST1 demonstrated elevated estrogen levels and exhibited precocious sexual maturation and increased fecundity. Female mice remained in estrus for prolonged periods and produced almost 50% more litters per mouse than wild-type mice over the same period of time. Thus, sulfate modification of the terminal glycosylation of LH plays a central role in regulating the hypothalamic-pituitary-gonad axis in vivo

Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand

We have constructed an artificial ligand for the hepatocyte-specific asialoglycoprotein receptor for the purpose of generating a synthetic delivery system for DNA. This ligand has a tetra-antennary structure, containing four terminal galactose residues on a branched carrier peptide. The carbohydrate residues of this glycopeptide were introduced by reductive coupling of lactose to the alpha- and epsilon-amino groups of the two N-terminal lysines on the carrier peptide. The C-terminus of the peptide, containing a cysteine separated from the branched N-terminus by a 10 amino acid spacer sequence, was used for conjugation to 3-(2-pyridyldithio)propionate-modified polylysine via disulfide bond formation. Complexes containing plasmid DNA bound to these galactose-polylysine conjugates have been used for asialoglycoprotein receptor-mediated transfer of a luciferase gene into human (HepG2) and murine (BNL CL.2) hepatocyte cell lines. Gene transfer was strongly promoted when amphipathic peptides with pH-controlled membrane-disruption activity, derived from the N-terminal sequence of influenza virus hemagglutinin HA-2, were also present in these DNA complexes. Thus, we have essentially borrowed the small functional domains of two large proteins, asialoglycoprotein and hemagglutinin, and assembled them into a supramolecular complex to generate an efficient gene-transfer system