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?E1,4GlcNAc?E1,2Man?D-, rather than the sequence Gal?E1,4GlcNAc?E1,2Man?D- that is found on many glycoproteins. The ?E1,4-N-acetylgalactosaminyltransferases (?EGTs) 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 Sia?D2,6GalNAc. 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 Sia?D2,6GalNAc are removed by the asialoglycoproteinreceptor. Using genetic strategies to alter the structure of the carbohydrates on LH, we have demonstrated that the precise structures 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 ?EGTs 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 ?EGTs 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 further define the impact of genetic ablation of the GalNAc-4-sulfotransferases on LH function as well as on kidney and brain functions. 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.
Many hormones such as luteinizing hormone are modified by the attachment of unique sugars that are critical for hormone activity. The sugars help determine how much estrogen and testosterone are produced at specific times. Understanding how synthesis of these sugars is regulated in mice will provide new insights into issues related to fertility, development, and susceptibility to hormonally sensitive cancers.
