title: Isolation and characterization of a glycosylated form of beta nerve growth factor in mouse submandibular glands.
abstract: 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.
authors.affiliation: Department of Anatomy and Cell Biology, University of Alberta, Edmonton, Canada.
authors.forename: R A
authors.initials: RA
authors.lastname: Murphy
authors.forename: V
authors.initials: V
authors.lastname: Chlumecky
authors.forename: L B
authors.initials: LB
authors.lastname: Smillie
authors.forename: M
authors.initials: M
authors.lastname: Carpenter
authors.forename: M
authors.initials: M
authors.lastname: Nattriss
authors.forename: J K
authors.initials: JK
authors.lastname: Anderson
authors.forename: J A
authors.initials: JA
authors.lastname: Rhodes
authors.forename: P A
authors.initials: PA
authors.lastname: Barker
authors.forename: K
authors.initials: K
authors.lastname: Siminoski
authors.forename: R B
authors.initials: RB
authors.lastname: Campenot
mesh_terms: 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*
chemical_list: D000596:Amino Acids; D009414:Nerve Growth Factors; D006026:Glycoside Hydrolases