Collagen, a major extracellular matrix macromolecule, is widely used for biomedical purposes. We investigated the absorption mechanism of low molecular weight collagen hydrolysate (LMW-CH) and its effects on osteoporosis in rats. When administered to Wistar rats with either [(14)C]proline (Pro group) or glycyl-[(14)C]prolyl-hydroxyproline (CTp group), LMW-CH rapidly increased plasma radioactivity. LMW-CH was absorbed into the blood of Wistar rats in the peptide form. Glycyl-prolyl-hydroxyproline tripeptide remained in the plasma and accumulated in the kidney. In both groups, radioactivity was retained at a high level in the skin until 14 days after administration. Additionally, the administration of LMW-CH to ovariectomized stroke-prone spontaneously hypertensive rats increased the organic substance content and decreased the water content of the left femur. Our findings show that LMW-CH exerts a beneficial effect on osteoporosis by increasing the organic substance content of bone.

By expression cloning using fluorescent-labeled high density lipoprotein (HDL), we isolated two clones that conferred the cell surface binding of HDL. Nucleotide sequence of the two clones revealed that one corresponds to scavenger receptor class B, type 1 (SRBI) and the other encoded a novel protein with 228 amino acids. The primary structure of the newly identified HDLbinding protein resembles GPI-anchored proteins consisting of an N-terminal signal sequence, an acidic region with a cluster of aspartate and glutamate residues, an Ly-6 motif highly conserved among the lymphocyte antigen family, and a C-terminal hydrophobic region. This newly identified HDL-binding protein designated GPI-anchored HDL-binding protein 1 (GPI-HBP1), was susceptible to phosphatidylinositol-specific phospholipase C treatment and binds HDL with high affinity (calculated K d ‫؍‬ 2-3 g/ml). Similar to SRBI, GPI-HBP1 mediates selective lipid uptake but not the protein component of HDL. Among various ligands for SRBI, HDL was most preferentially bound to GPI-HBP1. In contrast to SRBI, GPI-HBP1 lacked HDL-dependent cholesterol efflux. The GPI-HBP1 transcripts were detected with the highest levels in heart and, to a much lesser extent, in lung and liver. In situ hybridization revealed the accumulation of GPI-HBP1 transcripts in cardiac muscle cells, hepatic Kupffer cells and sinusoidal endothelium, and bronchial epithelium and alveolar macrophages in the lung.High density lipoprotein (HDL) 1 plays a key role in the transportation of cholesterol to extrahepatic tissues including steroidogenic tissues and in the reverse transportation of cholesterol from extrahepatic tissues to the liver (1). Unlike the low density lipoprotein (LDL) receptor pathway, the delivery of cholesterol from HDL to cells is mediated by selective lipid uptake from HDL particles and is independent of internalization of HDL. Reverse cholesterol transportation requires the extraction of cholesterol from extrahepatic cells by HDL and the subsequent delivery of cholesterol to hepatocytes.Several HDL-binding proteins have been identified including class B type I scavenger receptor (SRBI) (2, 3), two candidate hepatic HDL receptors designated HDL-binding proteins 1 and 2 (4, 5), 80-and 130-kDa GPI-anchored HDL-binding proteins expressed in human macrophages (6), 110-kDa GPI-anchored HDL-binding protein expressed in HepG2 cells (7), and recently characterized 95-kDa HDL-binding protein (8). To date, only SRBI appears to be a physiological HDL receptor based on the selective uptake of cholesterol esters into cells and the efflux of cholesterol from cells to HDL mediated by SRBI (1). Consistent with the postulated physiological role, SRBI is highly expressed in tissues that selectively take up cholesterol esters from HDL including liver, adrenal gland, testis, and ovary (3). Although hepatic overexpression of SRBI mediated by an adenovirus encoding SRBI resulted in a dramatic reduction of plasma cholesterol (9), the targeted disruption of the murine SRBI gene led to a modest inc...

Nucleotide sulfate, namely 3 -phosphoadenosine 5 -phosphosulfate (PAPS), is a universal sulfuryl donor for sulfation. Although a specific PAPS transporter is present in Golgi membrane, no study has reported the corresponding gene. We have identified a novel human gene encoding a PAPS transporter, which we have named PAPST1, and the Drosophila melanogaster ortholog, slalom (sll). The amino acid sequence of PAPST1 (432 amino acids) exhibited 48.1% identity with SLL (465 amino acids), and hydropathy analysis predicted the two to be type III transmembrane proteins. The transient expression of PAPST1 in SW480 cells showed a subcellular localization in Golgi membrane. The expression of PAPST1 and SLL in yeast Saccharomyces cerevisiae significantly increased the transport of PAPS into the Golgi membrane fraction. In human tissues, PAPST1 is highly expressed in the placenta and pancreas and present at lower levels in the colon and heart. An RNA interference fly of sll produced with a GAL4-UAS system revealed that the PAPS transporter is essential for viability. It is well known that mutations of some genes related to PAPS synthesis are responsible for human inherited disorders. Our findings provide insights into the significance of PAPS transport and post-translational sulfation.Glycosylation, phosphorylation, and sulfation are essential post-translational alterations of glycoproteins, proteoglycans, and glycolipids for normal growth and development. For sulfation, an activated form of sulfate, 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS), 1 is used as a common sulfate donor (1). Sulfate is transferred from PAPS to a defined position on the sugar residue by sulfotransferases. In higher organisms, PAPS is synthesized in the cytosol (2, 3) by a bifunctional PAPS synthetase having both ATP-sulfurylase and adenosine-phosphosulfate kinase activities (4). Since sulfation occurs in the lumens of the endoplasmic reticulum and Golgi apparatus (5), PAPS must be translocated from the cytosol into the Golgi lumen through a specific transporter localized in the microsomal membrane.Earlier studies had shown a saturable transport activity of PAPS using isolated Golgi vesicles (6) or reconstituted proteoliposome (7). To identify this transporter protein, the proteins responsible for PAPS translocating activity have been purified (8 -10). The characterization of these purified proteins revealed the kinetic behavior of a PAPS-specific transport through an antiport mechanism (8 -10); however, cloning of the transporter has not been reported.Recently, several nucleotide-sugar transporters (NSTs) have been cloned and characterized in mammals, yeast, protozoa, and plants (11-13). Nucleotide-sugars are the donor substrates for glycosylation, which is catalyzed by glycosyltransferases. These NST proteins are highly hydrophobic Type III multitransmembrane proteins localized in the Golgi or endoplasmic reticulum membrane and provide a specific substrate for the glycosylation. The structural conservation among NSTs has contributed to the identifi...

Sulfation is an important posttranslational modification associated with a variety of molecules. It requires the involvement of the high energy form of the universal sulfate donor, 3-phosphoadenosine 5-phosphosulfate (PAPS). Recently, we identified a PAPS transporter gene in both humans and Drosophila. Although human colonic epithelial tissues express many sulfated glycoconjugates, PAPST1 expression in the colon is trace. In the present study, we identified a novel human PAPS transporter gene that is closely related to human PAPST1. This gene, called PAPST2, is predominantly expressed in human colon tissues. The PAPST2 protein is localized on the Golgi apparatus in a manner similar to the PAPST1 protein. By using yeast expression studies, PAPST2 protein was shown to have PAPS transport activity with an apparent K m value of 2.2 M, which is comparable with that of PAPST1 (0.8 M). Overexpression of either the PAPST1 or PAPST2 gene increased PAPS transport activity in human colon cancer HCT116 cells. The RNA interference of the PAPST2 gene in the HCT116 cells significantly reduced the reactivity of G72 antibody directed against the sialyl 6-sulfo N-acetyllactosamine epitope and total sulfate incorporation into cellular proteins. These findings indicate that PAPST2 is a PAPS transporter gene involved in the synthesis of sulfated glycoconjugates in the colon.