A Heparin Binding Motif Rich in Arginine and Lysine is the Functional Domain of YKL-40

Abstract

The heparin-binding glycoprotein YKL-40 (CHI3L1) is intimately associated with microvascularization in multiple human diseases including cancer and inflammation. However, the heparin-binding domain(s) pertinent to the angiogenic activity have yet been identified. YKL-40 harbors a consensus heparin-binding motif that consists of positively charged arginine (R) and lysine (K) (RRDK; residues 144-147); but they don't bind to heparin. Intriguingly, we identified a separate KR-rich domain (residues 334-345) that does display strong heparin binding affinity. A short synthetic peptide spanning this KR-rich domain successfully competed with YKL-40 and blocked its ability to bind heparin. Three individual point mutations, where alanine (A) substituted for K or R (K337A, K342A, R344A), led to remarkable decreases in heparin-binding ability and angiogenic activity. In addition, a neutralizing anti-YKL-40 antibody that targets these residues and prevents heparin binding impeded angiogenesis in vitro. MDA-MB-231 breast cancer cells engineered to express ectopic K337A, K342A or R344A mutants displayed reduced tumor development and compromised tumor vessel formation in mice relative to control cells expressing wild-type YKL-40. These data reveal that the KR-rich heparin-binding motif is the functional heparin-binding domain of YKL-40. Our findings shed light on novel molecular mechanisms underlying endothelial cell angiogenesis promoted by YKL-40 in a variety of diseases.

Figure 1

YKL-40 constructs, mutants and heparin-binding property. (A) The schematic drawing of YKL-WT shows a signal peptide (SP) sequence, two chitinase domains between residues 90–142, one consensus heparin-binding RRDK motif between residues 144–147, and one putative KR-rich region between residues 334–345. Three bars indicate short peptides ySP1, ySP2 and ySP3, and bottom sequences represent YKL-WT and point mutation constructs. (B) Recombinant YKL-WT (1 μg) was used for a heparin-Sepharose affinity-binding assay. YKL-WT was eluted with 1 M NaCl (the last lane). The first lane indicates a positive YKL-WT control and the second lane contains the flow-through (FT). (C) Competitive binding assay with heparin. Recombinant YKL-WT was pre-mixed with heparin (0, 0.1, 1 or 10 μg/ml) for 30 min prior to introduction to the plate pre-coated with heparin for YKL-40 ELISA. Heparin-binding affinity was measured with absorbance at 405 nm. N = 4. *P < .05 compared with YKL-WT in the absence or presence of 0.1 μg/ml heparin. (D) ySP1, ySP2, or ySP3 (10 μg) was used for YKL-WT (1 μg) competitive heparin-binding assays. 1 M NaCl was used to elute final heparin-binding YKL-WT. A fraction of FT and wash buffer and whole concentrated elution were measured for YKL-WT (top blot) and the rest fractions of FT and wash buffer were concentrated and assayed for YKL-WT levels (bottom blot) using immunoblotting.

Figure 2

Lysine or arginine mutations of the C-terminal KR-rich domain led to decreased heparin-binding affinity of YKL-40. (A) Recombinant mutants (10 μg) were individually loaded onto a heparin-Sepharose column (0.5 ml). After washing, a concentration gradient of NaCl as indicated was applied to the column. All of flow through, wash buffer, and eluted fractions were used to measure YKL-40 levels. The first lane is recombinant YKL-WT as a positive control in immunoblotting. An additional elution with 1 M NaCl following the last elution did not show any YKL-40 remained in the column (data not shown). (B) Density of the bands shown in “A” was quantitatively analyzed using ImageJ software. The concentration of the protein was measured at each concentration of NaCl and was compared to the total cross-over protein level which was set up as 100%. N = 3. (C) Different mutants of YKL-40 were tested for their abilities to bind to heparin using an ELISA. Heparin binding of YKL-WT and individual mutants was dose-dependent. N = 3. *P < .05 compared with corresponding concentrations of YKL-WT.

Figure 3

Lysine or arginine mutations of the C-terminal KR-rich domain reduced the ability of mutant YKL-40 to compete with YKL-WT for heparin binding. (A) YKL-WT fused with GFP displayed a higher molecular mass than different mutant versions of YKL-40 in an immunoblotting assay, but are still recognized by rAY. (B) YKL-WT-GFP was preloaded onto each heparin-Sepharose column and after wash, different versions of YKL-40 (YKL-WT, K337A, K342A, R344A) as competitors from 1:1, 1:4, to 1:8 ratio were added to the columns and NaCl (0.5 M) was used for final elution.

Figure 4

Angiogenic activity of YKL-40 requires the presence of the lysine and arginine residues within the RK-rich domain. Recombinant YKL-WT, K337A, K342A, and R344A (200 ng/ml) were added to HMVECs in Matrigel. Tube formation was analyzed overnight (A). The tubes from in A were quantified (B). N = 3. *P < .05 compared with HMVECs treated with serum-free medium only. Different recombinant YKL-40 proteins were used for cell migration (C) and wound healing assays (D) as described in the Methods. N = 3–4 *P < .05 compared with HMVECs treated with serum-free medium only.

Figure 5

mAY, but not rAY, binds and blocks YKL-40 via its recognition of lysine and arginine residues within the KR-rich domain. (A) Equal amount of each recombinant YKL-WT, K337A, K342A, and R344A was subjected to immunoblotting using mAY and rAY. Coomassie blue staining was also used to demonstrate that there was equal samples loading. The intensity of the bound protein bands shown in immunoblotting using mAY was quantified. N = 3, *P < .05 compared with YKL-WT. (B) YKL-WT and R145G were subjected for immunoblotting using mAY and rAY. (C) Recombinant YKL-WT and R145G (200 ng/ml) were used to test tube formation of HMVECs. N = 3, *P < .05 compared with HMVECs treated with serum-free medium only as a control. (D) Equal amount of each recombinant YKL-WT, K337A, K342A, and R344A was immunoprecipated with either mAY or rAY followed by immunoblotting using either mAY or rAY. The first lane is the recombinant YKL-40 as a positive control in immunoblotting. One representative of quantified protein intensity from the top-three blots was shown. N = 3, *P < .05 compared with YKL-WT.

Figure 6

Pre-binding of mAY to YKL-40 blocks its heparin binding and mAY inhibits YKL-40 activity. (A) YKL-40 was pre-incubated with mIgG or mAY (1: 1 or 1: 10 molar ratio) overnight at 4°C. The complex samples were then loaded onto heparin-Sepharose columns as described in the Methods. Following wash, the eluted samples were used for immunoblotting. (B) The images shown in A were quantitatively analyzed. N = 3. *P < .05 compared with 1:1 mIgG, 1:1 mAY or 1:10 mIgG. The density of 1:1 mIgG flow through (FT) was set up as 1 unit. (C) HMVECs were used for cell migration assay as described in the Methods in the presence of mIgG (10 μg/ml, control), YKL-40 (200 ng/ml + mIgG), mAY (10 μg/ml), or rAY (10 μg/ml). N = 3, *P and +P < .05 compared with control and YKL-40, respectively.

Figure 7

Tumor development and angiogenesis induced by the breast cancer cell line MDA-MB-231 expressing ectopic different YKL-40 mutants. MDA-MB-231 cells were engineered to ectopically express different forms of YKL-40 mutants. After 48 h of incubation in serum-free medium, the expression of secreted YKL-40 proteins was measured using immunoblotting (A) or assayed for their ability to induce tube formation in HMVECs (B). These cells were transplanted into SCID/Beige mice as described in the Methods. Tumor volume was measured weekly and the final results at week 12 were quantified (C). Tumor samples were subjected to IHC with an anti-CD31 antibody (D) or rAY (E). Quantification of vessel density based on CD31 staining was analyzed using NIH ImageJ software. N = 5. *P and +P < .05 relative to controls. Bars: 100 μm.