Crystallization and characterization of human chorionic gonadotropin in chemically deglycosylated and enzymatically desialylated states.
Crystals suitable for X-ray diffraction studies at moderate resolution have been grown from two forms of human chorionic gonadotropin (hCG): HF-treated hCG and neuraminidase-treated hCG. The enzymatically desialylated form of hCG produced crystals that diffract to 2.8 A as compared to the HF-treated hCG crystals that diffract to 3.0 A. Although it was assumed that the high and heterogeneous carbohydrate content of the glycoprotein hormones inhibited their crystallization, this report suggests that it is the negatively charged surface sugars and neither the total carbohydrate content nor its heterogeneity which interferes with crystal formation. Chemical deglycosylation resulted in significantly increased protein degradation during crystal growth. Such peptide bond cleavages were observed to a much lesser extent in the crystals grown from neuraminidase-digested hCG. Sequence analysis of the HF-treated hCG crystals suggested that up to 45% of the molecules within the crystal had an acid-labile peptide bond cleaved. In contrast, the neuraminidase-treated hCG exhibited less than 9% of this type of cleavage. The increase in heterogeneity of the polypeptide chains within both crystals over that existent in the starting proteins was apparently due to changes occurring during crystal growth. The manner in which hCG was treated prior to crystallization was found to be a very important factor in the extent of peptide bond cleavages occurring during crystal growth. HF treatment of glycoproteins may render glycoproteins more susceptible to peptide bond cleavages during crystal growth.
Lustbader
J W
JW
Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Birken
S
S
Pileggi
N F
NF
Kolks
M A
MA
Pollak
S
S
Cuff
M E
ME
Yang
W
W
Hendrickson
W A
WA
Canfield
R E
RE
D000595:Amino Acid Sequence; D002241:Carbohydrates / Q000032:analysis; D006063:Chorionic Gonadotropin / Q000032:analysis / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006858:Hydrofluoric Acid / Q000494:pharmacology; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000378:metabolism; D014961:X-Ray Diffraction
D002241:Carbohydrates; D006063:Chorionic Gonadotropin; D009439:Neuraminidase; D006858:Hydrofluoric Acid