Biosynthesis and molecular cloning of sulfated glycoprotein 2 secreted by rat Sertoli cells

Abstract

Sulfated glycoprotein 2 (SGP-2) is the major protein secreted by rat Sertoli cells. Pulse-chase labeling shows that SGP-2 is synthesized as a cotranslationally glycosylated 64-kDa precursor that is modified to a negatively charged 73-kDa form before intracellular cleavage to the mature 47- and 34-kDa subunits. A plasmid cDNA library was constructed from immunopurified mRNA, and a recombinant clone containing the entire protein coding sequence of SGP-2 was isolated. The 1857-nucleotide cDNA consists of a 297-nucleotide 5' noncoding segment, a 1341-nucleotide coding segment, and a 219-nucleotide 3' noncoding sequence. The 5' noncoding region contains five ATG codons followed by four short open reading frames. The derived SGP-2 sequence has a molecular weight of 51,379 and contains six potential N-glycosylation sites. Proteolytic processing sites for the preproprotein were determined by amino-terminal sequencing of the isolated SGP-2 subunits. Northern blots show a wide tissue distribution for the 2.0-kb SGP-2 message, and computer sequence analysis indicates a significant relationship between SGP-2 and human apolipoprotein A-I.

Roles of clusterin in progression, chemoresistance and metastasis of human ovarian cancer

“…With the leader signal sequence, the 449 amino acid polypeptide is targeted to the endoplasmic reticulum (ER), further glycosylated, proteolytically cleaved into a-and b-subunits, held together by disulfide bonds and secreted outside the cell. 4,5 Depending on the degree of glycosylation, sCLU appears as 2 different protein bands by Western blot analyses: one full-length uncleaved 60 kDa protein, as well as another 80 kDa external sCLU, appears as 40 kDa a-and b-protein smears by SDS-PAGE under reducing conditions. 6 In contrast, the nuclear CLU (nCLU) protein was shown to be synthesized from a second in-frame AUG codon from an alternatively spliced nCLU mRNA, which eliminated Exon II (containing the first AUG and encoding the ER-targeting secretion signal sequence), and spliced Exons I and III together, placing the second in-frame AUG in Exon III as the first available translation start site.…”

“…20 This ambiguity between possible pro-and antiapoptotic activities of CLU has been addressed by many articles, 7,21 whereas its role in cell survival is still a matter of debate. Fortunately, the ability of the CLU gene to code for different/alternative protein isoforms was reported, 4,5,14 and it was hypothesized that there were 2 CLU protein isoforms generated in human cells. A secretory form (sCLU) is directed for secretion (prosurvival), and a nuclear form of the CLU protein (nCLU) is expressed in the cytoplasm and nucleus (proapoptotic).…”

Studies of specific gene induction during apoptosis of cell lines conditionally immortalized by SV40

“…10 mg of total RNA was fractionated on 1% agaroseformamide gel and transferred to nylon membrane (HybondN, Amersham) according to [10]. Probes for calmodulin, chondroitin sulfate proteoglycan core protein, clusterin, ubiquitin and GAPDH were obtained, respectively, by labeling of the insert of plasmid p21, p8.5 [11], pSP64-60HE [12], pHUB14.38 [13] and pGAPD-13 [14], respectively, with [32p]dCTP by random primers using the megaprime-labeling systems (Amersham). Probes for RP8, glutathione S-transferase (made from plasmids pRP-8 [15], pGST2 [16]) were labeled with [32p]dCTP by PCR of pBluescript inserts.…”

Tamoxifen Suppresses Clusterin Level through Akt Inactivation and Proteasome Degradation in Human Prostate Cancer Cells

“…It was first identified as a secreted glycoprotein in ram rete testis fluid enhancing cell aggregation in vitro (Blaschuk et al, 1983;Collard and Griswold, 1987). The primary product of the clusterin gene is a polypeptide of 449 amino acids and after proteolytic processing to remove the first 22 amino acids hydrophobic secretory signal sequence, clusterin is cleaved to produce its α and β subunits prior to secretion from the cells (Leskov et al, 2003).…”