PubMed · 8271963
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K F Nolan, K B Reid. 1993. Properdin.. https://doi.org/10.1016/0076-6879(93)23036-m
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The small ubiquitin-like modifier (SUMO) family in vertebrates includes three different family members that are conjugated as post-translational modifications to target proteins. SUMO-2 and -3 are nearly identical but differ substantially from SUMO-1. We used quantitative proteomics to investigate the target protein preferences of SUMO-1 and SUMO-2. HeLa cells were established that stably express His6-SUMO-1 or His6-SUMO-2. These cell lines and control HeLa cells were labeled with stable arginine isotopes, and His6-SUMOs were enriched from lysates using immobilized metal affinity chromatography. 53 SUMO-conjugated proteins were identified, including 44 novel SUMO targets. 25 proteins were preferentially conjugated to SUMO-1, 19 were preferentially conjugated to SUMO-2, and nine proteins were conjugated to both SUMO-1 and SUMO-2. SART1 was confirmed by immunoblotting to have both SUMO-1- and SUMO-2-linked forms at similar levels. SUMO-1 and SUMO-2 are thus shown to have distinct and overlapping sets of target proteins, indicating that SUMO-1 and SUMO-2 may have both redundant and non-redundant cellular functions. Interestingly, 14 of the 25 SUMO-1-conjugated proteins contain zinc fingers. Although both SUMO family members play roles in many cellular processes, our data show that sumoylation is strongly associated with transcription because nearly one-third of the identified target proteins are putative transcriptional regulators.
The cellular generation of proteoglycans with anticoagulant heparan sulfate (HSPGact) is determined by microsomal "HSact conversion activity" that functions in concert with the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to convert nonanticoagulant heparan sulfate (HSinact) to anticoagulant heparan sulfate (HSact) (Shworak, N. W., Fritze, L. M. S., Liu, J., Butler, L. D., and Rosenberg, R. D. (1996) J. Biol. Chem. 271, 27063-27071). Suspension cultures of L-33(+) cells in serum-free medium produce HSPGact and secrete HSact conversion activity. The secreted protein exhibiting HSact conversion activity was isolated by subjecting large volumes of conditioned suspension culture medium to heparin-AF Toyopearl affinity chromatography, Mono Q-FPLC, TSK SW3000-HPLC, and 3',5'-ADP-agarose affinity chromatography. The final product was purified approximately 700,000-fold relative to cellular material with a 5% overall recovery of HSact conversion activity. The isolated protein migrated on SDS-polyacrylamide gel electrophoresis as a broad band of Mr = 46,000 and co-migrated on nondenaturing acidic pH gel electrophoresis with HSact conversion activity. The purified component was identified as heparan sulfate D-glucosaminyl 3-O-sulfotransferase because it transferred sulfate from [35S]PAPS to the 3-O-position of D-glucosamine and D-glucosamine 6-O-sulfate of HSact precursor and HSinact precursor to produce nearly equivalent amounts of labeled HSact and HSinact. The exhaustive modification of wild-type LTA cell [35S]HS with either microsomal HSact conversion activity or purified enzyme increased HSact content from 9 to approximately 36%, which indicates that microsomal HSact conversion activity predominantly reflects the level of a 3-O-sulfotransferase that converts HSact precursor into HSact. The kinetic parameters of purified 3-O-sulfotransferase were determined for modification of HSact precursor and HSinact precursor. The apparent KM* and Vmax* with respect to PAPS concentration for sulfation of HSact precursor and HSinact precursor were 2.4 microM and 23 fmol of sulfate/min/ng of enzyme and 2.1 microM and 38 fmol of sulfate/min/ng of enzyme, respectively. There was substrate inhibition of the sulfation reaction at elevated HS concentration. The apparent KM* and Vmax* with respect to GAG concentration for sulfation of HSact precursor and HSinact precursor were 16 nM and 120 fmol of sulfate/min/ng of enzyme and 17 nM and 240 fmol of sulfate/min/ng of enzyme, respectively. The observation that purified 3-O-sulfotransferase catalyzes sulfation of HSact precursor and HSinact precursor in conjunction with a documented discordant regulation of 3-O-sulfate content in HSinact and HSact suggests that two discrete forms of the enzyme may exist.
A two-step chromatographic procedure for purification of basic fibroblast growth factor (bFGF) from high-cell-density cultures of recombinant E. coli is described. Heparin-Sepharose as a material which shows a high affinity to endothelial growth factors was used as sorbent for purification of bFGF from the soluble cell fraction. A one-step affinity chromatographic procedure resulted in very pure bFGF. However, this one-step affinity isolation of bFGF caused the loss of around 60% of the recombinant protein. A combination of ion-exchange chromatography with heparin-Sepharose affinity chromatography was favored for bFGF purification. A first cation-exchange chromatographic step resulted in a solution of bFGF with a purity of around 70%. The weak cation exchanger CM Sepharose C50 was preferred in comparison to the strong cation exchanger S-Sepharose because of the higher recovery of bFGF. With the ion-exchange chromatographic step prior to the heparin-Sepharose affinity chromatography, the total yield of recovery of bFGF increased to 56% compared to 40% using the one-step purification procedure with heparin-Sepharose. To characterize the equilibrium parameters of adsorption, batch experiments for the calculation of maximum capacities and dissociation constants for CM-Sepharose C50 and heparin-Sepharose were carried out. The equilibrium experiments revealed that adsorption of bFGF to the ion-exchange sorbent followed single-site interaction according to the Langmuir model of adsorption. The adsorption of bFGF to heparin-Sepharose was described by a double Langmuir approach of two independent binding sites with different maximum capacities and dissociation constants. The purified bFGF showed a high biological activity and circular dichroic spectra of a proper folded molecule. The analysis of the N-terminal amino acid sequence revealed a mixture of two fractions of bFGF, which both are characterized by the cleavage of the first amino acid methionine. In addition, half of the bFGF molecules lacked the second amino acid alanine.