Calcium buffering and protection from excitotoxic cell death by exogenous calbindin-D28k in HEK 293 cells.

Calbindin-D28k (CaBP) is a calcium-binding protein found in specific neuronal populations in the mammalian brain that, as a result of its proposed calcium-buffering action, may protect neurons against potentially harmful increases in intracellular calcium. We have stably transfected HEK 293 cells with recombinant human CaBP in order to determine the influence of this protein upon transient increases in intracellular ionic calcium concentration ([Ca(2+)](i)) induced either by transient transfection of the NR1 and NR2A subunits of the N-methyl-D-aspartate (NMDA) receptor and brief exposure to glutamate, photolysis of the caged calcium compound NP-EGTA, or exposure to the Ca(2+)]-ionophore 4-Br-A23187. The presence of CaBP did not significantly reduce the peak [Ca(2+)](i)stimulated by glutamate activation of NMDA receptors but significantly prolonged the recovery to baseline values. Flash photolysis of NP-EGTA in control cells resulted in an almost instantaneous increase in [Ca(2+)](i)followed by a bi-exponential recovery to baseline values. In cells stably expressing CaBP, the peak [Ca(2+)](i)levels were not statistically different from the controls, however, there was a significant prolongation of the initial portion of the slow recovery phase. In cells exposed to 4-Br-A23187, the presence of CaBP significantly reduced the rate of rise of [Ca(2+)](i), reduced the peak response, slowed the rate of recovery, and reduced the depolarization of mitochondria. In studies of delayed, Ca(2+)]-dependent cell death, CaBP transfected cells exhibited enhanced survival 24h after a 1-h exposure to 200 microM NMDA. However, necrotic cell death observed after the first 6h was not prevented by the presence of CaBP. These results provide direct evidence for a Ca(2+)-buffering effect of CaBP which serves to limit Ca(2+)entry and the depolarization of mitochondria, thereby protecting cells from death mediated most likely by apoptosis.

Factor X fusion proteins: improved production and use in the release in vitro of biologically active hirudin from an inactive alpha-factor-hirudin fusion protein.

Many recombinant proteins are synthesized as fusion proteins containing affinity tags to aid in the downstream processing. After purification, the affinity tag is often removed by using a site-specific protease such as factor Xa (FXa). However, the use of FXa is limited by its expense and availability from plasma. To develop a recombinant source of FXa, we have expressed two novel forms of FXa using baby hamster kidney (BHK) cells as host and the expression vector pNUT. The chimeric protein FIIFX consisted of the prepropeptide and the Gla domain of prothrombin linked to the activation peptide and protease region of FXa, together with a cellulose-binding domain (CBD(Cex)) as an affinity tag. A second variant consisted of the transferrin signal peptide linked to the second epidermal growth factor-like domain and the catalytic domain of FX and a polyhistidine tag. Both FX variants were secreted into the medium, their affinity tags were functional, and following activation, both retained FXa-specific proteolytic activity. However, the yield of the FIIFX-CBD(Cex) fusion protein was 10-fold higher than that of FX-CBD(Cex) and other forms of recombinant FX reported to date. The FXa derivatives were used to cleave two different fusion proteins, including a biologically inactive alpha-factor-hirudin fusion protein secreted by Saccharomyces cerevisiae. After cleavage, the released hirudin demonstrated biological activity in a thrombin inhibition assay, suggesting that this method may be applicable to the production of toxic or unstable proteins. The availability of novel FX derivatives linked to different affinity tags allows the development of a versatile system for processing fusion proteins in vitro.

Inhibition of calcium-dependent NMDA receptor current rundown by calbindin-D28k.

NMDA receptors are regulated by several different calcium-dependent processes. To determine if the presence of the intracellular calcium-binding protein calbindin-D28k can influence the calcium regulation of NMDA receptor activity, human embryonic kidney 293 cells were co-transfected with cDNAs for NMDA receptor subunits and calbindin. Recordings were made using the nystatin perforated patch technique to preserve intracellular contents. When compared with control cells (transfected with cDNA encoding beta-galactosidase in place of calbindin), the presence of calbindin had no effect on either calcium-dependent inactivation or the calcium-sensitive, time-dependent increase in glycine-independent desensitization of NMDA receptor-mediated currents. However, the development of calcium-dependent rundown of peak glutamate-evoked current was slowed significantly in calbindin versus beta-galactosidase co-transfected cells. This result was true for cells transfected with either NR1/NR2A or NR1/NR2B subunits, although calbindin was relatively less effective at inhibiting rundown in NR1/NR2B-expressing cells. NMDA peak current rundown has been attributed to calcium-induced depolymerization of the actin cytoskeleton. Therefore, our results indicate that although calbindin may not influence calcium-dependent regulatory processes occurring very near the NMDA receptor channel, it appears to be more effective at buffering local elevations in intracellular calcium at the actin cytoskeleton.