HCC-1, a novel chemokine from human plasma.

Abstract

SummaryA novel CC chemokine, HCC-1, was isolated from the hemoflhrate of patients with chronic renal failure. HCC-1 has a relative molecular mass of 8,673 and consists of 74 amino acids including four cysteines linked to disulfide bonds. HCC-1 cDNA was cloned from human bone marrow and shown to code for the mature protein plus a putative 19-residue leader sequence. Mature HCC-1 has sequence identity of 46% with macrophage inflammatory protein (MIP)-lc~ and MIP-][3, and 29-37% with the other human CC chemokines. Unlike MIP-I~x and the other CC chemokines, HCC-1 is expressed constitutively in several normal tissues (spleen, liver, skeletal and heart muscle, gut, and bone marrow), and is present at high concentrations (1-80 nM) in plasma. HCC-1 has weak activities on human monocytes and acts via receptors that also recognize MIP-lot. It induced intracellular Ca 2+ changes and enzyme release, but no chemotaxis, at concentrations of 100-1,000 nM, and was inactive on T lymphocytes, neutrophils, and eosinophil leukocytes. In addition, HCC-1 enhanced the proliferation of CD34 + myeloid progenitor ceils. It was as effective as MIP-lci, but about 100-fold less potent.H emofittration is a routine treatment for patients with chronic renal failure to remove substances that are normally cleared by the kidney. A filter membrane with a molecular mass cut-off of 20 kD is used, which virtually excludes plasma proteins (1, 2). Being available in large quantities, the hemoflltrate is an excellent source of biologically active human peptides that circulate in the blood. During the last 5 yr, a peptide bank was established from >200,000 liters of hemofiltrate, and several peptide hormones were purified (1). During the course of a systematic search for novel bioactive factors (3), we have identified a new member of the recently recognized family of chemotactic cytokines (chemokines). HCC-1 is structurally related to macrophage inflammatory protein (MIP)-loc Unlike MIP-lo~ and the other CC chemokines, HCC-1 is highly expressed in normal tissues and is present at high concentrations in human plasma. Materials and MethodsPurification. Peptides were extracted from batches of 2,000 liters of hemofiltrate by precipitation with 660 g/liter ammonium sulfate as described previously (2). The precipitate ("250 g) was dissolved in water (125 mg/ml). The peptides were precipitated again by addition of 4.5 vol of2-propanol, redissolved in 10 mM phosphate buffer, pH 3.0, and fractionated by cation exchange chromatography (Fractogel TSK SP-650 M, 6 • 20 cm; Merck, Darmstadt, Germany) with a 0-1.0-M NaC1 gradient in the same buffer. Fractions eluting at high salt concentrations were collected and further purified by preparative reverse-phase (lkP) HPLC (Parcosil RP C4, 300 ft,, 20-45 b~m, 3 • 12.5 cm; Biotek, Oestringen, Germany) using a gradient generated from 0.01 M HCI and 50% 2-propanol/30% methanol in 0.01 M HCI. Analytical R.P HPLC (Parcosil RP C4, 300 A, 5 btm, 1 • 12.5 cm, Biotek, Oestringen, Germany) was then performed using an acetonitrile/T...

Significance of N-Terminal Proteolysis of CCL14a to Activity on the Chemokine Receptors CCR1 and CCR5 and the Human Cytomegalovirus-Encoded Chemokine Receptor US28

“…Various analogs of CCL14a, including CCL14a(1-74), CCL14a(3-74), CCL14a(4 -74), and CCL14a(9 -74) have been described previously (16,19,20). In addition to these CCL14a analogs, we identified another variant, CCL14a(12-74), in human blood filtrate.…”

“…In addition to these CCL14a analogs, we identified another variant, CCL14a(12-74), in human blood filtrate. CCL14a(12-74) was identified in an isolation procedure for ligands of the chemokine receptor CCR5 using a CHO-K1 cell line expressing CCR5, G␣ 16 , and mitochondrial apoaequorin (20). Extraction of peptides from 10,000 L of human HF by cation exchange chromatography resulted in the generation of pH pool fractions that were used to establish a peptide library of circulating peptides by RP-HPLC.…”

“…Furthermore, an alternative splice variant of CCL14, which is referred to as CCL14b, carries an insert of 16 aa within the amino-terminal region of the sequence, which is introduced between aa 8 and 9 (17,18). Two unusual features of CCL14a are its constitutive expression in a large variety of tissues and its high plasma concentration, which ranges from 1.5 to 10 nM in normal human plasma (16). Whereas CCL14a(1-74) and its glycosylated form (19) represent inactive precursors, proteolytically activated CCL14a(9 -74) is a high affinity agonist of the chemokine receptors CCR1 and CCR5 and, to a lesser extent, of CCR3 (20).…”

“…The hemofiltrate (HF) CC chemokine CCL14a (HCC-1) is a member of the CC chemokine family and was originally isolated from the HF of patients with chronic renal failure (16). CCL14a consists of 74 aa, after cleavage of a 19-residue signal sequence, and shares a 46% sequence identity with CCL3 and CCL4.…”

Urokinase Plasminogen Activator and Plasmin Efficiently Convert Hemofiltrate CC Chemokine 1 into Its Active [9–74] Processed Variant

“…It is constitutively expressed by numerous tissues, leading to high plasma levels in normal subjects (ϳ10 nM). These levels may increase 10-fold in pathological conditions involving an inflammatory process (24). Full-size HCC-1 was shown to be a fairly weak agonist of CCR1 (25), and inactive on CCR5 (26).…”