title: Characterization of kallikrein-related peptidase 4 glycosylations.
abstract: Kallikrein-related peptidase 4 (KLK4) is a glycosylated serine protease that functions in the maturation (hardening) of dental enamel. Pig and mouse KLK4 contain three potential N-glycosylation sites. We isolated KLK4 from developing pig and mouse molars and characterized their N-glycosylations. N-glycans were enzymatically released by digestion with N-glycosidase F and fluorescently labeled with 2-aminobenzoic acid. Normal-phase high-performance liquid chromatography (NP-HPLC) revealed N-glycans with no, or with one, two, or three sialic acid attachments in pig KLK4 and with no, or with one or two sialic acid attachments in mouse KLK4. The labeled N-glycans were digested with sialidase to generate the asialo N-glycan cores that were fractionated by reverse-phase HPLC, and their retention times were compared with similarly labeled glycan standards. The purified cores were characterized by mass spectrometric and monosaccharide composition analyses. We determined that pig and mouse KLK4 have NA2 and NA2F biantennary N-glycan cores. The pig triantennary core is NA3. The mouse triantennary core is NA3 with a fucose connected by an α1-6 linkage, indicating that it is attached to the first N-acetyglucosamine (NA3F). We conclude that pig KLK4 has NA2, NA2F, and NA3 N-glycan cores with no, or with one, two, or three sialic acids. Mouse KLK4 has NA2, NA2F, and NA3F N-glycan cores with no, or with one or two sialic acids.
authors.affiliation: Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Eisenhower Place, Ann Arbor, MI 48108, USA.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Yamakoshi
authors.forename: Fumiko
authors.initials: F
authors.lastname: Yamakoshi
authors.forename: Jan C-C
authors.initials: JC
authors.lastname: Hu
authors.forename: James P
authors.initials: JP
authors.lastname: Simmer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003746:Dental Enamel Proteins / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007610:Kallikreins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D015394:Molecular Structure; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D034421:Sus scrofa
chemical_list: D003746:Dental Enamel Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D007610:Kallikreins; C120801:kallikrein 4; D019158:N-Acetylneuraminic Acid