A 40
kDa glycoprotein from dry secretion of sheep is implicated as a signaling factor and is named as SPS-40. This protein is secreted only during the early phase of involution when the drastic ...tissue remodeling occurs in the mammary gland. SPS-40 was purified from sheep dry secretions and crystallized using hanging drop vapour diffusion method. The crystals belong to orthorhombic space group
P2
12
12
1 with cell dimensions,
a
=
62.7
Å,
b
=
66.4
Å,
c
=
107.5
Å. The protein was also cloned for the determination of its complete amino acid sequence. The three-dimensional structure of SPS-40 was determined by X-ray crystallographic method at 2.0
Å resolution. The structure revealed the presence of an N-linked glycan chain at Asn39. The protein adopts a conformation with a classical (β/α)
8-barrel fold of triosephosphate isomerase (TIM) (residues 1–237 and 310–360) with an insertion of a small (α
+
β) domain (residues 240–307) similar to that observed in chitinases. However, the Leu substitution for Glu in the consensus catalytic sequence in SPS-40 causes a loss of chitinase activity. Furthermore, the sugar-binding groove in SPS-40 is distorted considerably from the standard chitin-binding site in chitinase enzymes and hence the binding of chitin-like oligosaccharides is considerably hampered. Three surface loops, His188–His197, Phe202–Arg212 and Phe244–Pro260 have exceptionally high values of
B-factors (average
=
70.5
Å
2), indicating the presence of a less defined region.
The crystal structure of a 40 kDa signalling glycoprotein from buffalo (SPB‐40) has been determined at 2.8 Å resolution. SPB‐40 acts as a protective signalling factor by binding to viable cells ...during the early phase of involution, during which extensive tissue remodelling occurs. It was isolated from the dry secretions of Murrah buffalo. It was purified and crystallized using the hanging‐drop vapour‐diffusion method with 19% ethanol as the precipitant. The protein was also cloned and its complete nucleotide and amino‐acid sequences were determined. When compared with the sequences of other members of the family, the sequence of SPB‐40 revealed two very important mutations in the sugar‐binding region, in which Tyr120 changed to Trp120 and Glu269 changed to Trp269. The structure showed a significant distortion in the shape of the sugar‐binding groove. The water structure in the groove is also drastically altered. The folding of the protein chain in the flexible region comprising segments His188–His197, Phe202–Arg212 and Tyr244–Pro260 shows large variations when compared with other proteins of the family.
A 40kDa glycoprotein from dry secretion of sheep is implicated as a signaling factor and is named as SPS-40. This protein is secreted only during the early phase of involution when the drastic tissue ...remodeling occurs in the mammary gland. SPS-40 was purified from sheep dry secretions and crystallized using hanging drop vapour diffusion method. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with cell dimensions, a=62.7A, b=66.4A, c=107.5A. The protein was also cloned for the determination of its complete amino acid sequence. The three-dimensional structure of SPS-40 was determined by X-ray crystallographic method at 2.0A resolution. The structure revealed the presence of an N-linked glycan chain at Asn39. The protein adopts a conformation with a classical (beta/alpha)(8)-barrel fold of triosephosphate isomerase (TIM) (residues 1-237 and 310-360) with an insertion of a small (alpha+beta) domain (residues 240-307) similar to that observed in chitinases. However, the Leu substitution for Glu in the consensus catalytic sequence in SPS-40 causes a loss of chitinase activity. Furthermore, the sugar-binding groove in SPS-40 is distorted considerably from the standard chitin-binding site in chitinase enzymes and hence the binding of chitin-like oligosaccharides is considerably hampered. Three surface loops, His188-His197, Phe202-Arg212 and Phe244-Pro260 have exceptionally high values of B-factors (average=70.5A(2)), indicating the presence of a less defined region.
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 P212121, with unit‐cell parameters a = 62.6, b = 67.4, c = 106.9 Å. 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 Å 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 (β/α)8‐barrel fold of triosephosphate isomerase (TIM barrel; residues 1–237 and 310–360) with the insertion of a small α+β 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.
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