The long term objective of this grant is to define the biological significance of a family of unique N-linked carbohydrate structures that contain the sequence GalNAc beta1,4GlcNAc beta1,2Manα-, rather than the sequence Gal beta1,4GlcNAc beta1,2Manα- that is found on many glycoproteins. The beta1,4-N-acetylgalactosaminyl-transferases ( betaGTs) that add GalNAc to this structure are protein-selective, recognizing specific amino acid sequences in the distinctive set of glycoproteins that become modified. When present in the substrate protein, the recognition sequences can increase the catalytic efficiency of GalNAc transfer to a carbohydrate acceptor 500 fold. The terminal GalNAc can remain unmodified or be substituted with SO4 to form terminal SO4-4-GalNAc or with Sialic acid (Sia) to form Siaα2,6GalNAc. We have shown that glycoproteins such as luteinizing hormone (LH) that bear SO4-4-GalNAc are removed from the blood by the Mannose/GalNAc-4-SO4-receptor while those bearing either GalNAc or Siaα2,6GalNAc are removed by the asialoglycoprotein-receptor. Using genetic strategies to alter the structure of the carbohydrates on LH, we have demonstrated that the precise structure of the carbohydrates determine the LH concentration in the blood and the amount of estrogen and testosterone that are produced. As a result sexual development and reproductive behavior are altered. We will characterize the recognition determinant in glycoprotein substrates that is utilized by the betaGTs via a novel new assay system that allows us to determine the efficiency of GalNAc addition both in vivo and in vitro. We will also examine how different domains or regions of the protein-selective betaGTs contribute to the selective addition of GalNAc to LH and other glycoproteins. Members of the family of GalNAc containing structures that we have characterized on LH and other pituitary glycoproteins are also found on glycoproteins produced in the brain, kidney, salivary glands, and other tissues. We will define the impact of genetic ablation of the GalNAc-4- sulfotransferases and the protein-selective betaGTs on LH function. Since we have shown that ablation of GalNAc-4-sulfotransferase has effect on reproduction, we expect that ablating one or both of the betaGTs that are protein-selective will have an even greater effect and reveal additional functions for these carbohydrates. We will also define regulation of the expression of the transferases that mediate the synthesis of this family of structures. Their expression may change over the course of the hormonal cycle and at specific times during sexual development such as when sexual maturity is attained. We have clearly shown that changing the structures of the carbohydrates on LH has a significant impact on its in vivo function. We have demonstrated that the pattern of glycosylation is modulated in vivo. This is one of the first instances in which modulation of carbohydrate structures has been shown to have an impact in vivo. Defining precisely how this is regulated is critical for understanding reproductive biology and for understanding the role of glycosylation in modulating the properties of a wide range of glycoproteins such as hormones, receptors, and matrix components.
We will define how the selective addition of a family of structurally unique carbohydrates to hormones that control sexual development and reproduction modulates the function of the hormones. The changes in hormone behavior have important implications for understanding reproduction and infertility. The impact on the levels of sex steroids produced over long periods may also have implications for the development of malignancies such as breast and prostate cancer that are hormone-dependent.
