Molecular cloning and expression of cDNA encoding a lumenal calcium binding glycoprotein from sarcoplasmic reticulum.

Abstract

Antibody screening was used to isolate a cDNA encoding the 160-kDa glycoprotein of rabbit skeletal muscle sarcoplasmic reticulum. The cDNA is identical to that encoding the 53-kDa glycoprotein except that it contains an in-frame insertion of 1308 nucleotides near its 5' end, apparently resulting from alternative splicing. The protein encoded by the cDNA would contain a 19-residue NH2-terminal signal sequence and a 453-residue COOH-terminal sequence identical to the 53-kDa glycoprotein. It would also contain a 436-amino acid insert between these sequences. This insert would be highly acidic, suggesting that it might bind Ca2 . The purified 160-kDa glycoprotein and the glycoprotein expressed in COS-1 cells transfected with cDNA encoding the 160-kDa glycoprotein were shown to bind 45Ca2+ in a gel overlay assay. The protein was shown to be located in the lumen of the sarcoplasmic reticulum and to be associated through Ca2+ with the membrane. We propose that this lumenal Ca2+ binding glycoprotein of the sarcoplasmic reticulum be designated "sarcalumenin."Muscle contraction is triggered by the release ofCa2l from the sarcoplasmic reticulum, whereas muscle relaxation is achieved by rapid reuptake of Ca2l from the cytosol into the lumen ofthe sarcoplasmic reticulum. Sequestration of Ca2l in the lumen of the sarcoplasmic reticulum is also an essential step in the overall contraction-relaxation cycle of muscle cells. The Ca2' pump has been found to be distributed along the membranes ofthe longitudinal sarcoplasmic reticulum and the nonjunctional region of the terminal cisternae (1), whereas the Ca+ release channel from sarcoplasmic reticulum has been localized in the junctional face of the terminal cisternae (1, 2). The low-affinity, high-capacity Ca2+ sequestering protein, calsequestrin, is located in the lumen of the terminal cisternae (3-5).The sarcoplasmic reticulum also contains two immunochemically related glycoproteins of unknown function, with apparent molecular masses of 53 and 160 kDa (6, 7). It has been proposed (8, 9) that the glycoproteins are involved in the regulation of Ca2+ transport. We have cloned cDNA encoding the 53-kDa glycoprotein (7) but were unable to deduce its function from analysis of its primary structure. In this study we describe the cloning of cDNA encoding the 160-kDa glycoprotein. ¶ Analysis of its deduced amino acid sequence and studies of its localization and Ca2" binding properties lead us to conclude that it is a Ca2+ binding protein located in the lumen of the longitudinal sarcoplasmic reticulum.MATERIALS AND METHODS Isolation, Analysis, and Expression of cDNA Clones. A Agtll cDNA expression library from rabbit fast-twitch skeletal muscle (a kind gift from

The endoplasmic reticulum-sarcoplasmic reticulum connection: distribution of endoplasmic reticulum markers in the sarcoplasmic reticulum of skeletal muscle fibers.

“…In particular, BiP is an ATPase specifically devoted to assist the correct folding of proteins and peptide loops exposed to the ER lumen (32), whereas calnexin has been shown to bind Ca2+ and proposed to play a role in the docking of specific lumenal proteins to the ER membranes (27). In the SR this putative function could concern sarcalumenin and the 53-kDa glycoprotein, two proteins that fail to express the KDEL sequence (10,11) and that therefore need an alternative mechanism to be retained. Of potentially even greater interest are the membrane proteins revealed by the anti-ER Ab in TC and JFM, especially the 28-kDa protein.…”

“…During their lifespan these proteins are transported to a pre-Golgi compartment, from which, however, they are retrieved to the ER after binding to a specific KDEL receptor (8). Ofthe SR lumenal proteins, CS (9) and other components-sarcalumenin (10, 11), 53-kDa glycoprotein (10,11), histidine-rich protein (12)-were found to lack the KDEL terminus. This, however, is not the case with two additional minor proteins, originally described as the high-affinity Ca2+ binding protein and the thyroid hormone binding protein and now recognized as calreticulin and protein disulfide isomerase (PDI), respectively (13,14).…”

Different transcription regulation routes are exerted by L- and D-amino acid enantiomers of peptide hormones

“…Sarco-endoplasmic reticulum (SR) calcium ATPases (SERCAs) are membrane-bound calcium pumps that hydrolyze ATP to pump calcium either across the plasma membrane or into the ER or SR (Sanyal et al, 2005). Sarcalumenin is a calcium-binding glycoprotein found only in the lumen of the SR (Leberer et al, 1989). Anoctamin is a membrane integral calciumaffected chlorine channel and may participate in osmoregulation (Milenkovic et al, 2010).…”

Identification and characterization of proteins in sarcoplasmic reticulum from normal and failing human left ventricles

“…The 53 000-and 160 000-Da glycoproteins of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum have been shown to be alternatesplice products of the same gene (Leberer et al, 1989b). Whether the same applies to the corresponding glycoprotein of cardiac muscle sarcoplasmic reticulum is uncertain, but the FIGURE 3.…”

“…Among these are a 105 000-Da Ca 2+ -transporting ATPase (Brandl et al, 1986(Brandl et al, , 1987; phospholamban, a 27000-29 000-Da pentamer whose phosphorylation is associated with stimulation of Ca 2+ uptake (Kirchberger et al, 1974;Tada et al, 1974); calsequestrin, a 55 000-Da protein that binds Ca 2+ within the sarcoplasmic reticulum with low affinity and high capacity ; two glycoproteins, 53000-Da and 130 000-Da, whose functions remain unknown but which may be involved in intraluminal Ca 2+ -binding and regulation of Ca 2+ uptake ; Campbell and M a c L e n n o n 1 9 8 1 , C a m p b e l l a n d Pepper et al, 1985;Helmke and Howard, 1987;Leberer et al, 1989a;Leberer et al, 1989b;Kutchai and Campbell, 1989); and a 400000-Da ryanodine-sensitive Ca 2+ channel (Lai et al, 1988;Innui et al, 1987;Campbell et al, 1987;Smith et al, 1988). The Ca 2+ -transporting ATPase of cardiac sarcoplasmic reticulum, like phospholamban, is the product of a gene expressed in cardiac and slow-twitch skeletal muscle but not in fast-twitch skeletal muscle (whose sarcoplasmic reticulum Ca 2+ ATPase is the product of a separate gene; Brandl et al, 1986).…”