Photoaffinity Labeling of a Cell Surface Polyamine Binding Protein

Abstract

Intracellular polyamine pools are partially maintained by an active transport apparatus that is specific for and regulated by polyamines. Although mammalian transport activity has been characterized by kinetic studies, the actual protein itself has yet to be identified, purified, or cloned. As one approach to this problem, we attempted photoaffinity labeling of plasma membrane proteins using two specifically designed and synthesized polyamine conjugates as photoprobes. The first is a spermidine conjugate bearing the photoreactive moiety 4-azidosalicylic acid at the N 4 position via an alkyl linkage, and the second is a norspermine conjugate with 4-azidosalicylic acid at the N 1 position via an acyl linkage. Labeling of murine L1210 lymphocytic leukemia cells was carried out at 4°C to promote selective alkylation of cell surface proteins. Separation of plasma membrane proteins from cells cross-linked with the N 4 -spermidine conjugate by SDS-polyacrylamide gel electrophoresis revealed two heavily labeled proteins at ϳ118 and ϳ50 kDa (designated p118 and p50, respectively). Band p118 was more well defined and much more intensely labeled. Analogous proteins were also observed in human U937 lymphoma cells. Specificity of labeling was strongly suggested by competition with polyamines and analogs during labeling and further indicated by the nearly identical labeling of the same protein by the N 1 -norspermine photoprobe but not by the unconjugated photoreagent. Neuraminidase pretreatment of L1210 cells increased mobility of the p118, suggesting that it was glycosylated and, thus, of plasma membrane origin. In transport-deficient L1210 cells, p118 and p50 were found to have a slightly higher molecular mass and were accompanied by a less distinct protein band (ϳ100 kDa). These findings indicate the presence of a polyamine binding protein at the surface of murine and human leukemia cells, which could be directly or indirectly related to the polyamine transport apparatus.

Role of endocytosis in the internalization of spermidine-C2-BODIPY, a highly fluorescent probe of polyamine transport

“…N%-(mercaptoethyl)spermidine, displayed a 60-fold higher K i value than did Spd-MANT. On the other hand, a derivative closely similar to Spd-MANT, but with a side arm shorter by one ethylene group, N%-(azidosalicylamidoethyl)spermidine, exhibited a 50-fold lower affinity than spermidine for the polyamine transporter in L1210 mouse leukaemia cells [12]. Therefore the thioethyl chain of Spd-C # -BODIPY might be less optimal than the aminopropyl group of the polyamine linker present in Spd-MANT, although the structural basis for these differences is unclear.…”

“…Conjugation of aromatic structures such as 4-azidosalicylic acid [12] and chlorambucil [13] to the polyamine core structure does not abrogate its strong binding to the polyamine carrier. This interesting property has recently been exploited for the design of fluorescent probes to study polyamine transport.…”

“…This property has previously been exploited for the biochemical characterization of the polyamine carrier. For instance, spermidine, spermine and norspermine were derivatized with "#&I-labelled 4azidosalicylic acid to generate photoreactive probes for the labelling of polyamine-binding proteins as potential candidates for the mammalian polyamine carriers [12]. The polyaminetransport system has also been used for the targeting of cytotoxic polyamine analogues [1] or chemotherapeutic agents conjugated to polyamines [13].…”

Selective labelling of cell-surface polyamine-binding proteins on leukaemic and solid-tumour cell types using a new polyamine photoprobe

“…Early attempts to purify the transporter protein by affinity chromatography relied on the interaction of positively charged polyamines with putative binding sites located on the cell surface [3]. A difficulty in this approach, however, has been the labile interactions of polyamines with the large number of negatively charged cell-surface proteins.…”

2,2′-Dithiobis(N-ethyl-spermine-5-carboxamide) Is a High Affinity, Membrane-impermeant Antagonist of the Mammalian Polyamine Transport System

“…Felschow et al (54) have described the specific labeling of discrete plasma membrane proteins, using 125 Ilabeled N 1 -azidosalicylamido-norspermine and N 4 -azidosalicylamidoethyl-spermidine as photoaffinity reagents. However, these conjugates are internalized by mammalian cells (54), and MESC-ASIB or similar derivatives could be useful as photoactivable probes to exclude labeling of intracellular proteins.…”