Biochemical and biologic characterization of murine monocyte chemoattractant protein-1. Identification of two functional domains.
Monocyte chemoattractant protein-1 (MCP-1) is a member of the chemokine-beta (or C-C) family of cytokines. Murine MCP-1, first identified as the JE gene, differs from human MCP-1 in molecular size and extent of glycosylation. We have used Chinese hamster ovary cells to express recombinant murine MCP-1 and find that its predominant form is a microheterogeneous protein of M(r) approximately 25,000. Most of MCP-1's microheterogeneity is due to variable amounts of sialic acid that are terminally attached to a constant number of O-linked oligosaccharide chains per molecule. This carbohydrate, along with a small amount of N-linked carbohydrate, accounts for 50% of the apparent molecular size of murine MCP-1 and is not required for in vitro monocyte chemoattractant activity. Mutational analysis shows that most of the carbohydrate is added to a 49-amino acid C-terminal domain that is not present in human MCP-1 and is not required for in vitro biologic activity, suggesting that murine MCP-1 consists of an N-terminal domain containing monocyte chemoattractant activity and a heavily glycosylated C-terminal domain of as yet unknown function. MCP-1 produced in COS cells contains a small amount of sulfate, but Chinese hamster ovary-produced MCP-1 does not. The absence of sulfate does not alter MCP-1's in vitro chemoattractant properties. In vitro, highly purified murine MCP-1 attracts monocytes, but not neutrophils, with a specific activity similar to human MCP-1 (EC50 approximately 0.5 nM). Equilibrium binding experiments with human monocytes reveal the presence of approximately 3000 binding sites per cell with a Kd of 0.77 nM. In vivo, injection of up to 1 micrograms murine MCP-1 in a variety of murine strains induces the appearance of a sparse mixed inflammatory infiltrate. The disparity between MCP-1's in vitro and in vivo effects suggests that other factors may be required to elicit a full-blown monocyte chemotactic response to MCP-1 in vivo.
Ernst
C A
CA
Department of Medicine, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115.
Zhang
Y J
YJ
Hancock
P R
PR
Rutledge
B J
BJ
Corless
C L
CL
Rollins
B J
BJ
D000818:Animals; D001483:Base Sequence; D018932:Chemokine CCL2; D002630:Chemotactic Factors / Q000737:chemistry*; D002634:Chemotaxis, Leukocyte; D017931:DNA Primers / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009000:Monocytes / Q000378:metabolism; D011994:Recombinant Proteins; D013431:Sulfates / Q000737:chemistry
D018932:Chemokine CCL2; D002630:Chemotactic Factors; D017931:DNA Primers; D006023:Glycoproteins; D011994:Recombinant Proteins; D013431:Sulfates