Isolation and characterization of a glycosylated form of beta nerve growth factor in mouse submandibular glands.
In the course of characterizing polyclonal antibodies to beta nerve growth factor (NGF) on immunoblot replicas of sodium dodecyl sulfate gels, we observed a protein (designated C protein) migrating as two bands (14.0 and 13.5 kDa) that copurifies with NGF and reacts strongly with its antibodies. The molecule is detectable in the 7 S, beta, and 2.5 S forms of NGF, accounting in the latter two for approximately 2% of total protein. The C protein can be separated from the A and B chains of beta-NGF on acetic acid-urea gels and on two-dimensional gels but not by isoelectric focusing alone. The molecule has been isolated to near purity on reversed-phase high performance liquid chromatography. Amino acid analyses and sequencing through 49 Edman cycles revealed that the protein preparation is composed of the intact and desoctapeptide (des-(1-8] polypeptide chains and suggested a glycosylation site at Asn-45. Following digestion with N-glycanase, the chains migrated on sodium dodecyl sulfate gels identically with the A and B chains of beta-NGF. Although this was accompanied by some degree of proteolytic degradation, the presence of glucosamine (approximately 4 mol/mol of single chain) was confirmed in acid hydrolysates on the amino acid analyzer. No amino sugars were detected in hydrolysates of the A chain nor was galactosamine recovered in either preparation. Glycosylated NGF promotes neuronal growth and survival in a manner indistinguishable from native 2.5 S NGF when tested in the chick sensory ganglion assay and with rat postnatal sympathetic neurons in a dissociated culture cell survival assay or in a compartmentalized culture growth assay. These studies reveal that NGF can be modified by glycosylation in a manner that does not reduce its biological activity.
Murphy
R A
RA
Department of Anatomy and Cell Biology, University of Alberta, Edmonton, Canada.
Chlumecky
V
V
Smillie
L B
LB
Carpenter
M
M
Nattriss
M
M
Anderson
J K
JK
Rhodes
J A
JA
Barker
P A
PA
Siminoski
K
K
Campenot
R B
RB
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases; D006031:Glycosylation; D007525:Isoelectric Focusing; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000302:isolation & purification*; D013363:Submandibular Gland / Q000032:analysis*
D000596:Amino Acids; D009414:Nerve Growth Factors; D006026:Glycoside Hydrolases