Back to Search
Start Over
A repeated triple lysine motif anchors complexes containing bone sialoprotein and the type XI collagen A1 chain involved in bone mineralization
- Source :
- The Journal of Biological Chemistry
- Publication Year :
- 2021
- Publisher :
- American Society for Biochemistry and Molecular Biology, 2021.
-
Abstract
- While details remain unclear, initiation of woven bone mineralization is believed to be mediated by collagen and potentially nucleated by bone sialoprotein (BSP). Interestingly, our recent publication showed that BSP and type XI collagen form complexes in mineralizing osteoblastic cultures. To learn more, we examined the protein composition of extracellular sites of de novo hydroxyapatite deposition which were enriched in BSP and Col11a1 containing an alternatively spliced "6b" exonal sequence. An alternate splice variant "6a" sequence was not similarly co-localized. BSP and Col11a1 co-purify upon ion-exchange chromatography or immunoprecipitation. Binding of the Col11a1 "6b" exonal sequence to bone sialoprotein was demonstrated with overlapping peptides. Peptide 3, containing three unique lysine-triplet sequences, displayed the greatest binding to osteoblastic cultures; peptides containing fewer lysine triplet motifs or derived from the "6a" exon yielded dramatically lower binding. Similar results were obtained with 6-carboxyfluorescein (FAM)-conjugated peptides and western blots containing extracts from osteoblastic cultures. Mass spectroscopic mapping demonstrated that FAM-peptide 3 bound to 90 kDa BSP and its 18 to 60 kDa fragments, as well as to 110 kDa nucleolin. In osteoblastic cultures, FAM-peptide 3 localized to biomineralization foci (site of BSP) and to nucleoli (site of nucleolin). In bone sections, biotin-labeled peptide 3 bound to sites of new bone formation which were co-labeled with anti-BSP antibodies. These results establish the fluorescent peptide 3 conjugate as the first nonantibody-based method to identify BSP on western blots and in/on cells. Further examination of the "6b" splice variant interactions will likely reveal new insights into bone mineralization during development.
- Subjects :
- 0301 basic medicine
Bone sialoprotein
collagen
Male
Nucleolus
Lysine
Peptide
confocal microscopy
Collagen Type XI
Biochemistry
bone
VR, variable region
Extracellular matrix
Column Buffer, 0.05 M sodium acetate buffer (pH 5.2) containing 8 M urea and 0.02% sodium azide
BSP, bone sialoprotein
CHAPS, (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate)
chemistry.chemical_classification
biology
Chemistry
RNA-Binding Proteins
Fluoresceins
BGP, β-glycerolphosphate
Research Article
N-terminal domain
Immunoprecipitation
extracellular matrix
Sialoglycoproteins
bone sialoprotein
PVDF, polyvinylfluoride
Bone and Bones
BMF, biomineralization foci
03 medical and health sciences
alternative splicing
Calcification, Physiologic
stomatognathic system
Extracellular
Animals
Integrin-Binding Sialoprotein
NTD, N-terminal domain
TBST, Tris-buffered saline containing Tween-20
Molecular Biology
cell culture
Osteoblasts
030102 biochemistry & molecular biology
Cell Biology
type XI collagen
Phosphoproteins
Molecular biology
Rats
030104 developmental biology
biology.protein
peptides
AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride
Npp, N-propeptide
Osteopontin
Nucleolin
FAM, 6-carboxyfluorescein
Subjects
Details
- Language :
- English
- ISSN :
- 1083351X and 00219258
- Volume :
- 296
- Database :
- OpenAIRE
- Journal :
- The Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....30688c227f718005597439415d47def5