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PNAS 98 (20): 11205-11210

Copyright © 2001 by the National Academy of Sciences.

Allurin, a 21-kDa sperm chemoattractant from Xenopus egg jelly, is related to mammalian sperm-binding proteins

John H. Olson*, Xueyu Xiang*, Tillmann Ziegert, Andrew Kittelson, Alan Rawls, Allan L. Bieber, and Douglas E. Chandler{dagger}

Molecular and Cellular Biology Program and the Departments of Biology and Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1501


Figure 1
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Figure 1 Sperm chemotaxis activity. Diffusible proteins from X. laevis egg jelly exhibit sperm chemoattractant activity. Sperm passage through a porous filter in a two-chamber assay was increased 5-fold over controls by the presence of soluble whole-egg jelly (SWEJ; 60 µg) or diffusible protein extract (12HEW; 30 µg), but not by "structural" (60 µg) egg jelly proteoglycans.

 

Figure 2
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Figure 2 FPLC purification of allurin. (A) Elution profile of 12HEW applied to a FPLC anion exchange column and eluted with 50 mM phosphate buffer (pH 7.45) and a linear gradient of NaCl. (B) Sperm chemotaxis activity. Sperm chemotaxis assays of each FPLC fraction indicate that high activity is found in fraction 3 with lower activity found in fraction 7. (C) SDS/PAGE analysis of each of the numbered fractions in A. Allurin is seen as a major band at Mr = 23 kDa (arrows) in fractions 3 and 7.

 

Figure 3
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Figure 3 HPLC purification of allurin. (A) HPLC purification of FPLC fraction 3. Elution profile of fraction 3 applied to a C-3 RP-HPLC column and eluted with acetonitrile. Allurin elutes as a sharp peak at 60% acetonitrile. SDS/PAGE analysis of HPLC-purified allurin is shown in the Inset (arrowhead). Protein standards at 200, 116, 97, 66, 55, 36, 31, 21, 14, and 6 kDa are shown in the lane to the left. (B) MALDI-TOF spectrum of HPLC peak. MALDI-TOF MS of HPLC-purified allurin. Single-, double-, and triple-charged molecular ions are seen at 21,053, 10,534, and 7,023 Da per electron unit, respectively. Cytochrome C, used as an internal standard, exhibits single- and double-charged molecular ions at 12,361 and 6,183 Da per electron unit, respectively. Because of matrix adduct-induced broadening of the single-charged molecular ion peak, the most accurate estimate of molecular mass is considered to be 21,068 Da, which is derived from the double-charged molecular ion peak. (C) Chemotaxis activity during purification. Dose-response curves for sperm chemoattractant activity in whole egg jelly ({lozenge}), 12HEW ({triangleup}), FPLC fraction 3 ({circ}), and HPLC-purified allurin ({square}). Data points represent means of 3 replicate experiments. All standard errors are less than 10% of the mean.

 

Figure 4
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Figure 4 Complete amino acid sequence of allurin and its alignment with mammalian TPX-1 and AEG sequences. Amino acid identity or conservative substitution at each position is shown in bold letters. Boxes enclose regions of high homology (>70%). Arrows point to positions of conserved cysteines. Regions of the allurin sequence that have been determined by chemical means (Edman degradation) are underlined, whereas those determined by molecular cloning are not. Only partial sequences for TPX-1 and AEG are shown. Sequence alignment was carried out by using CLUSTAL W for GenBank or EMBL/SwissProt accession nos. (top to bottom, respectively): ALLURIN; AAA40472.1/P16563; AAD48090.1/Q9R1L4; AAA61220.1/P16562; AAA37185.1/Q03401; AAB59716.1/P12020; and CAA64524.1/P54107.

 

Figure 5
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Figure 5 Phylogenetic analysis suggesting allurin is closely related to a vertebrate ancestral protein that gave rise to the mammalian CRISP family. The phylogenetic tree was constructed by using the nearest-neighbor joining method with MEGA 2 software (35) and the sequences of GenBank or EMBL/SwissProt accession nos. (top to bottom, respectively): CAA04729.1/O19010; CAA64527.1/P54108; AAC52616.1/Q60477; AAA61220.1/P16562; AAA40472.1/P16563; AAD48090.1/Q9R1L4; AAB59716.1/P12020; AAA37185.1/Q03401; CAA07160.1/NONE; AAD27611.1/Q9XSD3; CAA64524.1/P54107; ALLURIN; AAF54913.1/ Q9VFY2; AAA30333.1/P35786; AAB65434.1/O16135; AAA16207.1/ P35794; AAA16208.1/P35795; AAC25629/O82086; AAA03616.1/P04284; and CAA32228.1/P11670.

 


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