Structure of a bovine secretory signalling glycoprotein (SPC-40) at 2.1 Å resolution

Abstract

A recently discovered new class of 40 kDa glycoproteins forms a major component of the secretory proteins in the dry secretions of non-lactating animals. These proteins are implicated as protective signalling factors that determine which cells are to survive during the processes of drastic tissue remodelling. In order to understand its role in the remodelling of mammary glands, the detailed three-dimensional structure of the bovine signalling glycoprotein (SPC-40) has been determined using X-ray crystallography. SPC-40 was purified from bovine dry secretions and crystallized using the hanging-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 62.6, b = 67.4, c = 106.9 Angstrom. The protein was also cloned in order to determine its complete amino-acid sequence. Its three-dimensional structure has been determined using data to 2.1 Angstrom resolution. The amino-acid sequence determination of SPC-40 reveals two potential N-glycosylation sites at Asn39 and Asn345, but electron density for a glycan chain was only present at Asn39. The protein adopts a conformation with the classical (beta/alpha)(8)-barrel fold of triosephosphate isomerase (TIM barrel; residues 1-237 and 310-360) with the insertion of a small alpha+beta domain (residues 240-307) similar to that observed in chitinases. However, the substitution of Leu for Glu in the consensus catalytic sequence in SPC-40 caused a loss of chitinase activity. Furthermore, the chitin-binding groove in SPC-40 is considerably distorted owing to unfavourable conformations of several residues, including Trp78, Tyr120, Asp186 and Arg242. Three surface loops, His188-His197, Phe202-Arg212 and Tyr244-Pro260, have exceptionally high B factors, suggesting large-scale flexibility. Fluorescence studies indicate that various sugars bind to SPC-40 with low affinities.

Acute milk yield response to frequent milking during early lactation is mediated by genes transiently regulated by milk removal

“…Pathway analysis implicated decreased CHI3L-1 expression in the predicted reduction in cell death in response to 4ϫ milking. In agreement, CHI3L-1, which is a secreted protein, has been previously reported to be upregulated during mammary involution and remodeling in dairy cows (28,44), goats (29,40), and sheep (46). More recently, differential expression of CHI3L-1 mRNA was linked to lactogenic changes in mammary cells in culture (47).…”

Modification and periplasmic translocation of the biofilm exopolysaccharide poly-β-1,6- N -acetyl- d -glucosamine

“…Ala substitutions of conserved residues in the C-terminal domain of HmsF, the Yersinia pestis homolog of PgaB, had no effect on biofilm formation (33), further supporting the idea that PgaB 310-672 may be acting as a carbohydrate binding domain. Whereas the dissociation constant of PgaB 310-672 for β-1,6-(GlcNAc) 6 is weaker than those observed for chi-lectins, which have K d values for β-1,4-(GlcNAc) 6 (chitohexaose) in the low (<20 μM) range (26)(27)(28)(29)(30), stronger interactions may be observed with longer or partially de-N-acetylated PNAG oligomers. The simulation data support this notion because β-D-GlcNH 3 + density is only located along the electronegative groove of PgaB 310-672 , suggesting the domain may preferentially bind dPNAG ( Fig.…”

“…A number of GH18 members (chi-lectins) with mutations in the catalytic consensus motif (Fig. S2) retain the ability to bind chitin (26)(27)(28)(29)(30)(31)(32). Ala substitutions of conserved residues in the C-terminal domain of HmsF, the Yersinia pestis homolog of PgaB, had no effect on biofilm formation (33), further supporting the idea that PgaB 310-672 may be acting as a carbohydrate binding domain.…”

Crystal structure of a secretory signalling glycoprotein from sheep at 2.0 Å resolution

“…AY081150, Mohanty et al, 2003) and bovine (SPC-40) (GenBank Accession No. AY291312, Kumar et al, 2006). Similar proteins have been reported from human chondrocyte glycoprotein (HCGP-39) (GenBank Accession No.…”

“…It also shows a notable sequence identity to chitinases (Renkema et al, 1995) and chitinase-like protein (Sun et al, 2001;Tsai et al, 2004). The structure of SPS-40 is homologous to that of MGP-40 and SPC-40 (Kumar et al, 2006;Mohanty et al, 2003) as well as to that of HCGP-39 (Fusetti et al, 2003;Houston et al, 2003). In spite of overall similarities, SPX-40 proteins diVer in the folding properties of its several parts including shape of the socalled sugarbinding groove, the organization of the Xexible domain and the conformation of its glycan chain.…”