Purification of Guinea Pig YKL40 and Modulation of Its Secretion by Cultured Articular Chondrocytes

J Cell Biochem. 1998 Jun 15;69(4):414-24. doi: 10.1002/(sici)1097-4644(19980615)69:4<414::aid-jcb3>3.0.co;2-q.

Abstract

The aim of this study was to purify, characterize, and study the regulation at the chondrocyte level of the guinea pig (gp) homologue of human (R) YKL40, a putative marker of arthritic disorders. Studying YKL40 in guinea pigs is of particular interest, as age-related osteoarthritis develops in this species spontaneously. Both N-terminal sequencing and total amino acid composition of gpYKL40 purified from the secretion medium of cultured articular chondrocytes indicate a high degree of identity with hYKL40. gpYKL40 was found to contain complex N-linked carbohydrate, as demonstrated by N-glycosidase F and endoglycosidase F digestion. Isoelectric focusing demonstrated the presence of a major band at pI 6.7. The secretion of gpYKL40 by confluent articular chondrocytes in the extracellular medium was studied by immunoblotting. gpYKL40 was released by chondrocytes continuously over a 7 day period and did not appear to be degraded by proteinases, as its signal intensity in cell-free medium at 37 degrees C did not decrease with time. Thus, gpYKL40 displays high stability and accumulates in extracellular medium without reaching a steady-state level. Among the main factors known to regulate cartilage metabolism, IL-1beta, TNF-alpha, bFGF, or 1,25(OH)2D3 did not alter the basal level of gpYKL40, and retinoic acid had a slight inhibitory effect; TGF-beta and IGF-I and -II dose-dependently and inversely modulated this basal level. TGF-beta at 5 ng/ml decreased extracellular gpYKL40 2.9-fold, whereas IGF-I and IGF-II at 50 ng/ml increased extracellular gpYKL40 3.6- and 3.4-fold, respectively. The present biochemical and biological findings give new insights for studying the function of YKL40 in cartilage.

MeSH terms

  • Adipokines
  • Amino Acid Sequence
  • Amino Acids / analysis
  • Animals
  • Cartilage, Articular / metabolism*
  • Cells, Cultured
  • Chitinase-3-Like Protein 1
  • Chondrocytes / metabolism*
  • Cytokines / pharmacology
  • Glycoproteins*
  • Glycosylation
  • Guinea Pigs
  • Isoelectric Point
  • Kinetics
  • Lectins
  • Molecular Sequence Data
  • Molecular Weight
  • Proteins / chemistry
  • Proteins / isolation & purification*
  • Proteins / metabolism*
  • Sequence Analysis

Substances

  • Adipokines
  • Amino Acids
  • CHI3L1 protein, human
  • Chitinase-3-Like Protein 1
  • Cytokines
  • Glycoproteins
  • Lectins
  • Proteins