1.3 Identification of pFGE: paralog of FGE
Data base searches of the human genome revealed a coding region highly similar to FGE (48% sequence identity, 62% similarity), which was designated as SUMF2 and the protein as paralog of FGE (pFGE) [Cosma et al., 2003;Dierks et al.,2003;
Landgrebe et al., 2003]. In human, SUMF2 gene is located on chromosome 7q11.
Deuterostomia, including vertebrates and echinodermata, also express a paralog of FGE. Gene duplication of a common SUMF ancestor has obviously occurred at the level of a single exon gene after the evolution of insects and before that of deuterostomia [Landgrebe et al., 2003]. pFGE along with FGE belongs to a large protein family currently comprising 164 members sharing the common domain of unknown function DUF323.
1.3.1 Characteristic features of pFGE
1.3.1.1 Expression and localization of endogenous and recombinant pFGE pFGE like FGE is ubiquitously expressed. A single SUMF2 transcript of 2.0-2.1 kb is detectable by Northen blot analysis of polyA+ RNA from various tissues and of total RNA from skin fibroblast (Fig. 1.4A) [Mariappan et al.,2005]. The expression pattern is quite similar to that of SUMF1 encoded FGE. Relative to β-actin RNA, the abundance of SUMF2RNA varies by one order of magnitude, being highest in pancreas, kidney and lowest in brain.
The nucleotide sequence of human pFGE predicts a protein of 301 residues.
An N-terminal signal peptide (residues 1-26, Fig. 1.10) directs translocation of the nascent polypeptide into the lumen of endoplasmic reticulum. Indirect im-munofluorescence of endogenous pFGE in human skin fibroblasts revealed it to be in the lumen of endoplasmic reticulum colocalizing with the ER marker protein PDI (Fig. 1.9A). However, recombinant pFGE with C-terminal HA or His tag showed co-localization not only with PDI but also with GM130, a Golgi marker protein (Fig. 1.9B and C). This was substantiated by immunoelectron microscopy, which showed localization of pFGE in the ER and Golgi stacks, sparsely in endosomes and lysosomes [Mariappan et al.,2005]. However, a small fraction of endogenous pFGE escapes from the ER into secretions. Overexpression of pFGE results in its massive secretion [Mariappan et al.,2005].
10 Chapter1. Introduction
PDI
PDI pFGE
pFGE-HA
pFGE-HA
Merge
Merge
Merge GM 130
A
B
C
Figure 1.9: Subcellular localization of endogenous and C-terminally tagged pFGE.
Indirect immunofluorescence of pFGE (red) and PDI or GM130 (green). The merge reveals the co-localization of PDI and endogenous pFGE (A) and C-terminally tagged pFGE-HA either with PDI (B) or with GM130 (C). A and B [Mariappan et al.,2005], C: Mariappan M., unpublished data.
1.3.1.2 Structural properties of pFGE
pFGE has a single N-glycosylation site at Asn191. Intracellular pFGE was shown to have a high mannose type oligosaccharide, which becomes processed during secretion to hybrid and complex type structures containing fucose and sialic acid residues.
The phylogenetic sequence analysis ofSUMF2encoded pFGE has revealed that pFGE has three highly conserved regions forming three subdomains of a DUF323 do-main (Fig. 1.10), analogous to FGE (seeFig. 1.6). In pFGE these subdomains make up more than 85% of the molecule. Digestion with elastase, a serine proteinase, or thermolysin, a zinc proteinase, generated two stable fragments by cleavage within the short linker sequence connecting the first and second subdomain. However, the crystal structure of pFGE revealed a novel fold with a strikingly low degree of secondary structure, as it has been determined for FGE (see Section 1.2.4).
The tertiary structure is characterized by an asymmetric partitioning of secondary structure elements and is stabilized by two calcium cations and a disulphide bond
1.3. Identification of pFGE: paralog of FGE 11
Figure 1.10: pFGE domain structure. Phylogenetic sequence conservation analysis of pFGE has revealed three highly conserved subdomains (I, II and III). SP represents the cleavage site for signal peptide, ‘C’ represents the cysteines which are involved in disulfide bonding and ‘N’
represents glycosylation site at Asn191.
between Cys156 and Cys290 (Fig. 1.11) [Dickmanns et al., 2005]. This disulfide bond is also conserved in FGE, underscoring its stabilizing function. However, pFGE lacks other cysteine residues which are found in FGE. pFGE structure was used to determine the crystal structure of FGE, revealing a high structural similarity [Dierks et al.,2005].
pFGE and FGE structures represent the first three-dimensional models of a
Oligosaccharide
Figure 1.11: Crystal structure of pFGE. Secondary structural elements showing two Ca2+
ions (red spheres), part of oligosaccharide sidechain at N191 (sticks) and disulfide bond (yellow stick) between C156 and C290 [Dickmanns et al.,2005].
12 Chapter1. Introduction DUF323 domain containing protein. Due to the close relation of pFGE to the well-characterized FGE, the novel fold of the DUF323 domain is denoted as ”FGE” fold.
In addition, the structure point to a possible cellular function of pFGE. Indeed, the active site cleft in FGE showed a high similarity to pFGE. Many of the residues (Ala149, Ser155, Trp299, Ser356, Asn360 and Leu361) lining the substrate-binding groove in FGE are conserved in pFGE (Fig. 1.12). The crystal structure also predicts the possibility of a pFGE/FGE dimer. In fact pFGE but not FGE formed a homodimer in the asymmetric unit of the crystal.
pFGE FGE
Figure 1.12: Comparison of the peptide binding cleft of pFGE and FGE. Left: pFGE in gray with the cleft (greenish yellow) most likely to be involved in the binding of a sulfatase polypeptide chain. Pro120, colored in blue, corresponds to Pro182 of FGE that has been shown to cross-link to a photoaffinity labeled substrate peptide. Right: the FGE cleft is shown in greenish yellow. The residues different to pFGE are highlighted in red and are predominantly located in the region of the active centre, close to the catalytically active cysteines 336 and 341 which are absent in pFGE [Dickmanns et al.,2005].
1.3.1.3 Probable biological role for pFGE
Localization, carbohydrate processing, and secretion upon overexpression as well as the protease sensitivity of the linker region between the first two subdomains and the general structure are the properties that pFGE shares with FGE. Moreover, the relative abundance of their RNAs in different tissues is rather similar. In addition, by photoaffinity labeling and yeast two-hybrid assay it was demonstrated that pFGE shows interaction with ASA derived peptides [Mariappan et al., 2005]. All these observations suggested that pFGE and FGE fulfill similar functions. But so far, no
1.4. ER retention mechanisms 13