Jeffrey Y. Su

Cystatin F Is a Glycosylated Human Low Molecular Weight Cysteine Proteinase Inhibitor

A previously undescribed human member of the cystatin superfamily called cystatin F has been identified by expressed sequence tag sequencing in human cDNA libraries. A full-length cDNA clone was obtained from a library made from mRNA of CD34-depleted cord blood cells. The sequence of the cDNA contained an open reading frame encoding a putative 19-residue signal peptide and a mature protein of 126 amino acids with two disulfide bridges and enzyme-binding motifs homologous to those of Family 2 cystatins. Unlike other human cystatins, cystatin F has 2 additional Cys residues, indicating the presence of an extra disulfide bridge stabilizing the N-terminal region of the molecule. Recombinant cystatin F was produced in a baculovirus expression system and characterized. The mature recombinant protein processed by insect cells had an N-terminal segment 7 residues longer than that of cystatin C and displayed reversible inhibition of papain and cathepsin L (K i = 1.1 and 0.31 nm, respectively), but not cathepsin B. Like cystatin E/M, cystatin F is a glycoprotein, carrying two N-linked carbohydrate chains at positions 36 and 88. An immunoassay for quantification of cystatin F showed that blood contains low levels of the inhibitor (0.9 ng/ml). Six B cell lines in culture secreted barely detectable amounts of cystatin F, but several T cell lines and especially one myeloid cell line secreted significant amounts of the inhibitor. Northern blot analysis revealed that the cystatin F gene is primarily expressed in peripheral blood cells and spleen. Tissue expression clearly different from that of the ubiquitous inhibitor, cystatin C, was also indicated by a high incidence of cystatin F clones in cDNA libraries from dendritic and T cells, but no clones identified by expressed sequence tag sequencing in several B cell libraries and in >600 libraries from other human tissues and cells.

Development of a human stanniocalcin radioimmunoassay: serum and tissue hormone levels and pharmacokinetics in the rat.

Stanniocalcin (STC) is a polypeptide hormone that was first discovered in fish and recently identified in humans and other mammals. In fish STC is produced by one gland, circulates freely in the blood and plays an integral role in mineral homeostasis. In mammals, STC is produced in a number of different tissues and serves a variety of different functions. In kidney, STC regulates phosphate reabsorption by proximal tubule cells, whereas in ovary it appears to be involved in steroid hormone synthesis. However there is no information on circulating levels of STC in mammals or the regulation of its secretion. In this report we have developed a radioimmunoassay (RIA) for human STC. The RIA was validated for measuring tissue hormone levels. However human and other mammalian sera were completely devoid of immunoreactive STC (irSTC). To explore the possibility that mammalian STC might have a short half-life pharmacokinetic analysis was carried out in rats. STC pharmacokinetics were best described by a two compartment model where the distribution phase (t1/2(alpha)) equaled 1 min and the elimination phase (t1/2(beta)) was 60 min. However the STC in the elimination phase no longer crossreacted in the RIA indicating it had undergone substantial chemical modification, which could explain our inability to detect irSTC in mammalian sera. When we compared the pharmacokinetics of human and fish STC in mammalian and fish models the human hormone was always eliminated faster, indicating that human STC has unique structural properties. There also appears to be a unique clearance mechanism for STC in mammals. Hence there are major differences in the delivery and biology of mammalian STC. Unlike fishes, mammalian STC does not normally circulate in the blood and functions instead as a local mediator of cell function. Future studies will no doubt show that this has had important ramifications on function as well.

High Expression of Stanniocalcin in Differentiated Brain Neurons

Stanniocalcin (STC) is a glycoprotein hormone first found in fish, in which it regulates calcium homeostasis and protects against hypercalcemia. Human and mouse stc cDNA were recently cloned. We found a dramatically upregulated expression of STC during induced neural differentiation in a human neural crest-derived cell line, Paju. Immunohistochemical staining of sections from human and adult mouse brain revealed abundant presence of STC in the neurons with no activity in the glial cells. STC expression was not seen in immature brain neurons of fetal or newborn mice. Given that STC has been found to regulate calcium/phosphate metabolism in some mammalian epithelia, we suggest that STC may act as a regulator of calcium homeostasis in terminally differentiated brain neurons.