CD90

Thy-1 or CD90 is a 25–37 kDa heavily N-glycosylated, glycophosphatidylinositol (GPI) anchored conserved cell surface protein with a single V-like immunoglobulin domain, originally discovered as a thymocyte antigen. Thy-1 can be used as a marker for a variety of stem cells and for the axonal processes of mature neurons. Structural study of Thy-1 lead to the foundation of the Immunoglobulin superfamily, of which it is the smallest member, and led to the first biochemical description and characterization of a vertebrate GPI anchor.

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Discovery and Nomenclature

Thy-1 was discovered in early 1960s during the search for heterologous antisera against mouse leukemia cells. It was originally named theta (θ) antigen, then Thy-1 (THYmocyte differentiation antigen 1) due to its prior identification in thymocytes (precursors of T cells in the thymus). The human homolog was isolated in 1980 as a 25kDa protein (p25) of T-lymphoblastoid cell line MOLT-3 binding with anti-monkey-thymocyte antisera.^[1].

The conserved gene and its alleles

Thy-1 has been conserved throughout vertebrate evolution and even in some invertebrates, with homologs described in many species like squid, frogs, chickens, mice, rats, dogs, and humans.

The Thy-1 gene is located at human chromosome 11q22.3 (mouse chromosome 9qA5.1). Some believe that there may be a functional significance of both this gene and CD3 delta subunit (T3D) mapping to chromosome 11q in man and chromosome 9 in mouse, though there is no homology (in fact this speculation lead to its localization in chromosome 11q - the human chromosome region syntenic to mouse chromosome 9 which harbored T3D). In mouse there are two alleles Thy1.1 (Thy 1a, CD90.1) and Thy1.2 (Thy 1b, CD90.2) which differ by one amino acid at position 89 (arginine in Thy-1.1 and glutamine in Thy-1.2). Thy 1.2 is expressed by most strains of mice, whereas Thy1.1 is expressed by some like AKR/J and PL mouse strains.

The Protein

The 25-kDa core protein (excluding the heavy glycosylation) of rodent Thy-1 is 111 or 112 amino acids in length, and is N-glycosylated at three sites (In contrast to only two glycosylation sites for human Thy-1). The 162aa (murine, 161 for human) Thy1 precursor has 19 amino acid (aa 1-19) signal sequence and 31 amino acid (aa 132-162) C-terminal transmembrane domain that is present in pro form but removed when transferring the 112 amino acid (aa 20-131) mature peptie to GPI anchor which would attach through the aa 131.

Some of the common monoclonal antibodies used to detect this protein are clones OX7, 5E10, K117 and L127. There have been some reports of Thy1 monoclonal antibodies cross reacting with some cytoskeletal elements: anti Thy-1.2 with actin in marsupial, murine, and human cells and anti Thy-1.1 with vimentin, and were suggested to be due to sequence homology by studies done more than 20 years back.^[2]

Thy-1, like many other GPI anchored proteins can be shed by special types of Phospholipase C eg PI-PLC (phosphatidyl-Inositol Phospholipase C, or PLC β). it can also be involved in cell to cell trasfer of GPI anchored proteins like CD55 and CD59.

Expression

Thy1 expression varies between species. Amongst the cells reported to generally express Thy-1 areThymocytes (precursor of T cells in the Thymus) & CD34(+) prothymocytes; neurons, mesenchymal stem cells, hematopoietic stem cells, NK cells, murine T-cells, endothelium(mainly in High endothelial venules or HEVs where diapedesis takes place), renal glomerular mesangial cells, circulating metastatic melanoma cells, Follicular Dendritic Cells (FDC), a fraction of fibroblasts and myofibroblasts.

Detailed expression of Thy-1

• In mice, Thy-1 is also found on thymocytes, peripheral T cells, myoblasts, epidermal cells, and keratinocytes. It is one of the so called famous "pan T cell markers"(of mice) like CD2, CD5 and CD28.
• In humans, Thy-1 is also expressed by endothelial cells, smooth muscle cells, a subset of CD34+ bone marrow cells, and umbilical cord blood- and fetal liver-derived hemopoietic cells.

• Thy-1 is present on a fraction of brain cells and a fraction of fibroblasts of most vertebrate species studied.
• Nervous tissue: Thy-1 expression in the nervous system is predominantly neuronal, but some glial cells also express Thy-1 especially at later stages of their differentiation. One study compared Thy-1 expression in four human neuronal cell lines, two neuroglial cell lines, and fresh tumor cells of neuronal origin and found three of the four neuronal cell lines, all of the neuroglial cell lines, and 80% of the tumors to be strongly positive for Thy-1.^[3] Brain part specific ELISA reports are available which show highest concentrations of Thy1 protein in the striatum and hippocampus, followed by the neocortex, cerebellum, spinal cord, and the retina and optic nerve. Thy1 promoter has often been assumed to be "brain specific". "Neuron specific" mouse thy1 promoter has been used to drive "brain specific" forced expression of proteins eg mutated Amyloid precursor protein(APP) as transgenic animal models of Alzheimer's disease[1].^[4] Thy-1 expression in the brain is developmentally regulated. Thy-1 levels in the neonatal rat brain, as well as the developing human brain, are low compared to adult brain. During the first few weeks of postnatal development, Thy-1 levels increase exponentially as the brain matures.
• Lymphoid tissue Thy-1 expression is highly variable between species. In humans, Thy-1 expression is restricted to only a small population of cortical thymocytes^[5]and not expressed in mature human T cells.^[6]. It is probably the most abundant glycoprotein of murine thymocytes, with about One million copies per cell covering up to 10–20% of the cell surface.^[7] Mouse cortical thymocytes express higher levels of Thy-1 than medullary thymocytes which in turn express more than lymph node cells (~200,000 copies/cell). A similar inverse developmental temporal expression profile is seen in rats T cells, although rat Thy-1 is lost at an earlier stage of T cell maturation.^[8] Thy-1 is expressed only expressed on thymocytes in rats (contrast to thymocytes and splenocytes in mice). The third intron of the mouse Thy-1 gene has a 36 base pair region that recruits nuclear transcription factors, such as Ets-1-like NF, expressed in thymocytes and splenocytes. The homologous region of the rat gene lacks the Ets-1-like NF binding site, but instead binds another NF expressed in rat thymocytes but not splenocytes.

Induction of Thy-1 expression

• Agents shown to induce Thy1 expression include: Thymopoietin, thymosin, prostaglandins, nerve growth factor, IL-1, TNF, PMA, Ca2+ ionophore, and diacylglycerol (DAG)^[9]

Localization

As a GPI-anchored protein, Thy-1 is present in the outer leaflet of lipid rafts in the cell membrane. In case of neurons it is known to be expressed strongly in the mature axon. Axon hillock (neuronal equivalent of tight junction) can act as a barrier for its lateral spread even though it has no transmembrane segment. Thy-1 has been suggested to interact with G inhibitory proteins, the Src family kinase (SFK) member c-fyn, and tubulin within lipid rafts. In rats and mice, Thy-1 protein is present on the soma (cell body) and dendrites of neurons but is not expressed on axons until axonal growth is complete, and is again temporarily suppessed during axonal injury. HIV-1 Matrix co-localizes with Thy-1 in lipid rafts, the site of virus particle budding from cells, and Thy-1 is incorporated into virus particles as a result of this process.

Glycosylation

Thy-1 is one of the most heavily glycosylated membrane proteins with a carbohydrate content up to 30% of its molecular mass.^[10] The composition of Thy-1 carbohydrate moieties varies considerably between different tissues or even among cells of the same lineage at different stages of differentiation: eg, galactosamine only in brain Thy-1, sialic acid in thymic Thy-1 in far excess than brain Thy-1, that too increasing in parallel with T cell maturation

Function

The function of Thy-1 has not yet been fully elucidated. It has speculated roles in cell-cell and cell-matrix interactions, with implication in neurite outgrowth, nerve regeneration, apoptosis, metastasis, inflammation, and fibrosis.

Role in cognition

The Thy-1 knockout (KO) mice are viable and appear grossly normal. They display normal social interactions and normal learning in a maze, but fail to learn from social cues (e.g. learning from other mice which foods are safe to eat as compared to wild-type mice). This failure can be rescued by the transgenic expression of Thy-1 or pharmacologic treatment with a GABA (A) receptor antagonists. This suggests that Thy-1 KO mice have excessive GABAergic inhibition in the dentate gyrus and regional inhibition of long-term potentiation.

Axon growth regulation

Crosslinking anti-Thy-1 Ab can promote neurite outgrowth which is dependent on G{alpha}i and L- and N-type calcium channel activation. The ligand for promotion of neurite outgrowth on astrocytes is not yet identified, but the inhibitory ligand has been suggested to be integrins. Thy1 is one of the known ligands of beta 3 integrins. Interaction of thy1 expressed on maturing axons with beta 3 integrins expressed on mature astrocytes may be the cause of haulting of axon growth.

T-cell activation

Crosslinking Thy-1 molecules in the membrane raft, in the context of strong costimulatory signaling through CD28 in mouse T cells can act to some extent as a substitute activating signal for T-cell receptor signaling. Conversely it can substitute CD28 costimulation for activation through the TCR.[2]

Apoptosis/Necrosis

Cross linking antibody induced aggregation of Thy1 cause death of thymocytes and mesangial cells mainly by apoptosis despite Bcl2 upregulation. The death of mesangial cells seems to be apoptosis by TUNEL staining or annexin V staining, but electron microscopy suggest it is necrosis.

Antibody target for animal model of Glomerulonephritis

Single tail vein intravenous injection of antibody (OX7 mouse monoclonal IgG) against Thy1.1 in rats is used as a standard animal model to produce experimental mesangioproliferative glomerulonephritis^[11] which is popularly known in the field of nephrology as antiThy1 GN.

Tumor suppression

It has also been proven to be a tumor suppressor for some tumors.^[12] It probably is aided by its action in upregulating thrombospondin, SPARC (osteonectin), and fibronectin. However it has also been speculated to aid in extravasation in circulating melanoma cells. In case of prostate cancer it has been shown to be expressed in cancer associated stroma but not in normal stroma and has been suggested to be of potential help for cancer specific drug targeting [3].

Role in cell adhesion, extravasation, migration

Acting through several integrins and probably a few yet unknown other receptors Thy-1 mediates adhesion of leukocytes and monocytes to endothelial cells and fibroblasts, melanoma cells to endothelium, and thymocytes to thymic epithelium. ^[13]. Thy1 expression comes on when endothelial cells are activated. It has been shown to interact with the leukocyte integrin Mac1 (CD11b/CD18) and may play a role in leukocyte homing and recruitment. ^[14]

Modulating fibrosis

Role of Thy-1 in fibrosis and fibroblast differention may have some tissue variation. In lung fibrosis Thy-1 level is suppressed in stimulated fibroblasts. Thy1 knock out mice have increased fibrosis in the lung. Fibrosis induced by radiation mimicking chemotherapeutic agent Bleomycin is also increased in these mice.

Other roles

Thy-1 knock out mice also show impaired cutaneous immune responses and abnormal retinal development: thinning of the inner nuclear, inner plexiform, ganglion cell, and outer segment layers of the retina.

Use in stem cell biology

Thy-1 can be considered as a surrogate marker for various kind of stem cells (e.g. hematopoietic stem cells or HSCs). It is one of the popular combinatorial surface markers for FACS for stem cells in combination with other markers like CD34. During sorting, mouse HSCs fall under Thy1^hi subpopulation, while human HSCs fall under the Thy1^lo subpopulation. In humans, Thy-1 is only expressed on neurons^[15]. Thy 1 is also a marker of other kind of stem cells, for example: Mesenchymal stem cells, Hepatic stem cells ("Oval cells").^[16], Keratinocyte stem cells ^[17], putative endometrial progenitor/(?)stem cells ^[18]

References