CD137L Structure and Expression

CD137L is a 34 kD type II membrane glycoprotein with a carboxy-terminal extracellular domain and its gene is on chromosome 17 in the mouse (Goodwin et al., 1993). Although human CD137 ligand (huCD137L) is present in both T and B cells of the peripheral blood, the ligand is preferentially expressed in primary B cells and B cell lines. Daudi, a B cell lymphoma, is one of the B cell lines with the highest number of ligand molecules (Zhou et al., 1995). Scatchard analysis gave a kd of 1.4 x 10-9 M and number of ligand molecules per Daudi cell of 4.2 x 103. Primary B cells when stimulated with pokeweed mitogen showed enhanced ligand expression. On the other hand, the ligand for murine CD137 is present at low levels on T cell lines (non-activated and anti-CD3-activated), pre-B cell lines and a number of immature macrophage cell lines. Also, CD137 AP (a fusion protein consisting of the extracellular domain of CD137 fused to human placental alkaline phosphatase) exhibited no binding to a glial tumor cell line, HeLa cells, or COS cells. On the other hand, anti-IgM-activated primary B cells showed higher binding of CD137 AP than anti-CD3-activated primary T cells. Scatchard analysis indicated that the A20 B lymphoma cells had 3680 binding sites per cell with a kd of 1.86 nM, and Western analysis showed that CD137L has a molecular mass of approximately 18-25 kD. Chalupny et al. (1992) reported that a fusion of the extracellular domain of CD137 with the Fc portion of human IgG1 bound to various extracellular matrix proteins. However, we and others have identified a high affinity ligand (CD137L) (Pollok et al., 1994; Goodwin et al., 1993) and cloned it (Goodwin et al., 1993). Consistent with its original assignment to the TNFR family, CD137L was found to be homologous to members of the emerging family of type II transmembrane glycoproteins that are counter-receptors to members of the TNFR superfamily (Armitage, 1994; Smith et al., 1994).

6. CD137L Gene

Accession number: human (NM00381) and mouse (NM009494) The human CD137L gene (accession number: NM003811) (Figure 1.3A) is made up of three exons for the coding region, and two introns in which there is one exon for the 5' UTR. The mouse CD137L gene (accession number: NM009404) (Figure 1.3B), on the other hand, is composed of three exons and two introns.

7. CD137L Protein

Accession numbers: human (AI357267, BM790119, U03398, NM003811) and mouse (NM009404)

CD137L is a 34 kD glycoprotein with probable involvement of the N-linked sites, and possibly also the three putative O-linked sites (Goodwin et al., 1993). Under reducing conditions, CD137L has an apparent MW of ~97 kD suggesting that it is a disulfide-linked homodimer. The C terminal 200 residues of full-length recombinant CD137L appear to contain glycosylation sites and at least one interchain disulfide bond capable of generating homodimers. This region has the lowest degree (14-16%) of sequence identity with various other family members. Based on sequence data, CD137L has a tertiary structure very similar to that of TNF and LT- a, which is consistent with its being oligomeric. The region between strands D

Figure 1.3. Genomic organization of human (A) and murine (B) CD137 ligands. Exons are shown as boxes; untranslated regions (UTR) are represented by black shading, and protein coding regions by no shading; they are labeled with Roman numerals. Lengths of introns and exons are shown in Arabic numerals (base pairs).

Figure 1.3. Genomic organization of human (A) and murine (B) CD137 ligands. Exons are shown as boxes; untranslated regions (UTR) are represented by black shading, and protein coding regions by no shading; they are labeled with Roman numerals. Lengths of introns and exons are shown in Arabic numerals (base pairs).

and I is not well conserved and can only be loosely aligned using secondary structure prediction techniques. There are two conserved residues, Leu164 and Trp166, in the loop between B-strands B and B'. These residues immediately precede a segment of the loop, residues Gln169 through Ala172, which is important for the activity of TNF (Goh and Porter, 1990). The hydrophobic side-chains of Leu164 and Trp166 are buried and appear to anchor this important loop in the structure.

The C-terminal residues of TNF and LT-a together form p-strand I, an integral part of the tertiary fold. There are seven extra C-terminal residues in CD137L; these probably form a flexible tail that does not exist in other family members. This putative tail is reminiscent of the N-terminal tails of soluble TNF and LT-a (9 and 25 residues, respectively) that modulate the biological activities of these cytokines (Goh and Porter, 1990). Cysteine placement varies across the ligand family. Of the three cysteines in the extracellular domain of CD137L, the second and third, both in loops at the same end of the b-sandwich, are well positioned to form a disulfide bond. The first cysteine (Cys137), nine residues N-terminal to the homologous region, is thus probably the cysteine involved in the homodimer link.

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