A comprehensive proteomics and genomics analysis reveals novel transmembrane proteins in human platelets and mouse megakaryocytes including G6b-B, a novel immunoreceptor tyrosine-based inhibitory motif protein

Abstract

The platelet surface is poorly characterized due to the low abundance of many membrane proteins and the lack of specialist tools for their investigation. In this study we identified novel human platelet and mouse megakaryocyte membrane proteins using specialist proteomics and genomics approaches. Three separate methods were used to enrich platelet surface proteins prior to identification by liquid chromatography and tandem mass spectrometry: lectin affinity chromatography, biotin/NeutrAvidin affinity chromatography, and free flow electrophoresis. Many known, abundant platelet surface transmembrane proteins and several novel proteins were identified using each receptor enrichment strategy. In total, two or more unique peptides were identified for 46, 68, and 22 surface membrane, intracellular membrane, and membrane proteins of unknown subcellular localization, respectively. The majority of these were single transmembrane proteins. To complement the proteomics studies, we analyzed the transcriptome of a highly purified preparation of mature primary mouse megakaryocytes using serial analysis of gene expression in view of the increasing importance of mutant mouse models in establishing protein function in platelets. This approach identified all of the major classes of platelet transmembrane receptors, including multitransmembrane proteins. Strikingly 17 of the 25 most megakaryocyte-specific genes (relative to 30 other serial analysis of gene expression libraries) were transmembrane proteins, illustrating the unique nature of the megakaryocyte/platelet surface. The list of novel plasma membrane proteins identified using proteomics includes the immunoglobulin superfamily member G6b, which undergoes extensive alternate splicing. Specific antibodies were used to demonstrate expression of the G6b-B isoform, which contains an immunoreceptor tyrosine-based inhibition motif. G6b-B undergoes tyrosine phosphorylation and association with the SH2 domain-containing phosphatase, SHP-1, in stimulated platelets suggesting that it may play a novel role in limiting platelet activation.

Fig. 1

Comparison of proteins isolated by WGA affinity chromatography, biotin/NeutrAvidin (NA) affinity chromatography and free flow electrophoresis (FFE). A, platelet whole cell lysate (WCL) and proteins isolated by the three enrichment techniques were resolved on 4-20% SDS-PAGE gels and stained with Colloidal Coomassie blue. Bands corresponding to αIIb, β3, actin and GPIbβ were identified by tandem mass spectrometry and are shown to the left of the panels. WGA, wheat germ agglutinin affinity chromatography; biotin/NA, biotin/NeutrAvidin affinity chromatography; FFE-PM, free flow electrophoresis-plasma membrane fraction; FFE-IM, free flow electrophoresis-intracellular membrane fraction. Images shown are representative of three WGA, three biotin/NA, and two FFE enrichment experiments. B, aliquots taken at various stages of the WGA affinity chromatography procedure, including elution by N-acetylglucosamine (GlcNac), were western blotted for PECAM-1 and GPIbα.

Fig. 2

MS/MS spectra of G6b peptides. Peptides corresponding to each MS/MS spectra are shown in the top right corner of each panel, along with the G6b isoforms from which each peptide may have been derived, the Swiss-Prot/TrEMBL accession number (in parenthesis), and the experiment and band slice identification numbers. The start and end of each peptide are indicated by dots. Adjacent amino acids to the peptide identified are also included (outside dots). Selected b- and y-ions identified are indicated. (A) Peptide TVLHVLGDR is present in all seven isoforms of G6b. (B) Pepetide LPPQPIRPLPR is only present in G6b-A. (C) Peptide IPGDLDQEPSLLYADLDHLALSR is present in G6b-B, -C and -E.

Fig. 3

Expression of G6b in human platelets. A, (i) whole cell lysates were prepared from human platelets and HEK 293T cells transiently transfected with either plasmid alone (mock) or a G6b-B expression plasmid (G6b-B) were western blotted for G6b-B using a rabbit anti-G6b-B polyclonal antibody raised against two peptides from the cytoplasmic tail of the protein; (ii) as a control, the same samples western blotted in (i) were blotted with pre-immune serum from the same rabbit in which the G6b-B anti-serum was raised. B, G6b-B undergoes an increase in tyrosine phosphorylation in response to CRP and thrombin stimulation, and interacts with SHP-1 in human platelets. G6b-B was immunoprecipitated (IP) from whole cell lysates prepared from resting platelets and platelets stimulated with either 10 μg/mL CRP or 5U/mL thrombin. Samples were western blotted for tyrosine phosphorylated proteins, then stripped and blotted for G6b-B, followed by SHP-1. C, G6b-B is tyrosine phosphorylated in resting and CRP and thrombin activated platelets and interacts with SHP-1. SHP-1 was immunoprecipitated from whole cell lysates prepared from resting platelets and platelets stimulated with either 10 μg/mL CRP or 5 U/mL thrombin. Samples were western blotted for tyrosine phosphorylated proteins, then stripped and blotted for G6b, followed by SHP-1. Results are representative of three experiments.

Fig. 4

The ploidy of bone marrow-derived megakaryocytic cells was assessed by flow cytometry in the presence of propidium iodide. Mature megakaryocytes (>64n) were used to generate the SAGE library.