Biochemical characterization of recombinant and circulating human Spα

Abstract

Human Spalpha is a soluble protein expressed by macrophages present in lymphoid tissues (spleen, lymph node, thymus, and bone marrow), for which little functional and structural information is available. It belongs to the group B of the scavenger receptor cysteine-rich superfamily (SRCR-SF) that includes the lymphocyte surface receptors CD5 and CD6 among others. Spalpha is able to bind to different cells of the immune system (monocytes and lymphocytes), which suggests that it may play an important role in the regulation of this system. To study Spalpha, an episomal mammalian expression system (pCEP-Pu/HEK 293-EBNA) was used to produce a recombinant form (rSpalpha) that was utilized for biochemical studies and for the generation of specific hybridomas. Four monoclonal antibodies were selected for their reactivity against rSpalpha by Western blot, immunoprecipitation, and enzyme-linked immunosorbent assays. The monoclonal antibodies recognized three different epitopes on Spalpha. The monoclonal antibodies revealed the existence of two Spalpha isoforms of 38 and 40 kDa, resulting from different sialic acid content. They also showed that Spalpha is a relatively abundant serum protein (60 micro g/ml) that mostly circulates in association with other serum proteins. Accordingly, rSpalpha allowed affinity chromatography isolation of polyclonal and monoclonal immunoglobulin M (IgM). These data indicate that Spalpha is a circulating protein that may play a role in the homeostasis of IgM antibodies.

CD6 binds to pathogen-associated molecular patterns and protects from LPS-induced septic shock

“…The recombinant proteins were expressed in HEK 293-EBNA and affinity-purified as reported (12,37). Their purity was assessed by SDS/PAGE and staining with Coomassie blue.…”

Recent advances in blood‐related proteomics

“…4A), and was found as being strongly associated with IgM. CD5L is a soluble protein expressed in lymphoid tissues (spleen, lymph node, thymus, and bone marrow), for which little functional and structural information is available [51]. It belongs to group B of the scavenger receptor cysteinerich superfamily, which includes the lymphocyte surface receptors CD5 and CD6 amongst others.…”

The macrophage soluble receptor AIM/Api6/CD5L displays a broad pathogen recognition spectrum and is involved in early response to microbial aggression

“…21 Briefly, AIM/Api6/CD5L cDNA was obtained by reverse transcription of mouse peritoneal exudate macrophage mRNA isolated using the Ultraspec RNA isolation system (Biotecx Laboratories, Houston, TX, USA) from healthy C57Bl/6 mice and subsequent polymerase chain reaction amplification with the 59-GCCCGGCTAGC-GTCTCCAACCAAAGTGCAG-39 (forward) and 59-CGCG-GATCCTCACACATCAAAGTCTGTGCA-39 (reverse) AIMspecific primers, which incorporated NheI and BamHI restriction sites (underlined), respectively. The amplified polymerase chain reaction product (1013 bp) was cloned into appropriately restricted pCEP-Pu/BM-40 vector such that the AIM protein sequence starting at Ser23 (from the immature unprocessed protein) and ending at Val351 was fused in frame just after the BMP-40 signal peptide in the final construct.…”

“…For microbial binding assays, aliquots of ,5310 7 bacteria and ,5310 6 fungi were incubated o/n at 4 uC under gentle orbital rotation with different amounts of biotin-labelled rAIM in Tris-buffered saline (TBS) plus 3% BSA and 5 mM CaCl 2 to a final volume of 0.5 ml, in the presence or absence of different competitors (LPS and LTA at 0, 10, 50 or 100 mg, and rSpa at 0, 0.5, 1 and 2 mg). Following incubation, microbial cells were pelleted and washed once with 0.5 ml of TBS plus BSA and Ca 21 and twice with 0.5 ml of TBS alone to remove nonspecifically bound proteins. The washed pellets were resuspended in reducing Laemmli's sample buffer for 15 min at 100 uC and separated on 8% SDS-PAGE gels, followed by electrotransfer to a nitrocellulose membrane (BioRad, CA, USA).…”

“…20 However, differences in the glycosylation and tissue expression patterns of these two proteins could account for functional discrepancies between them. 18,20 As secreted molecules, they are readily detected (at ng mg/ml level) in human and mouse serum 20,21 with Spa levels varying in different clinical situations. 16,[22][23][24][25] RNA analysis indicates that both molecules are expressed in lymphoid tissues (thymus, spleen fetal liver, bone marrow), but a more detailed immunohistochemical analysis on AIM/Api6/CD5L expression showed that it is restricted to only a fraction of tissue macrophages.…”