Amino-terminal processing of cell surface heparin-binding epidermal growth factor-like growth factor up-regulates its juxtacrine but not its paracrine growth factor activity.

Human heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) expressed on Chinese hamster ovary (CHO) cells is synthesized as a 19-kDa major, and 22- and 27-kDa minor, membrane-anchored precursors (proHB-EGF). In contrast, the 27-kDa species is major and the 19- and 22-kDa ones are minor in mouse proHB-EGF. The juxtacrine growth factor activities of human and mouse proHB-EGFs on CHO cells toward EP170.7 cells in co-culture are significantly different. To investigate the relationship between the juxtacrine growth factor activities and the molecular species, we prepared human-mouse chimeras. Chimeras that have the human amino-terminal sequence with a mouse EGF-like domain showed approximately 8-fold up-regulation of the juxtacrine growth factor activity and the predominance of a 19-22-kDa major species. In contrast, chimeras that have the mouse amino-terminal sequence with a human EGF-like domain showed approximately 5-fold down-regulation of the juxtacrine activity and the predominance of the 27-kDa major species. A Gly32·HB-EGF (117-amino acid form), which is amino-terminally extended, induced the same mitogenic activity as that of Arg73·HB-EGF (75-amino acid form), which is amino-terminally truncated. These results strongly suggested that amino-terminal processing of human proHB-EGF would be required for up-regulation of its juxtacrine growth factor activity, but not for its paracrine activity.

The analysis of the nucleotide sequence of human HB-EGF cDNA predicts a precursor protein of 208 amino acids composed of a putative signal peptide and heparin-binding, EGF-like, transmembrane, and cytoplasmic domains (1)(2)12). The HB-EGF precursor can be cleaved on the plasma membrane to yield a biologically active protein comprising 75-87 amino acids (2,13,14). While soluble HB-EGF is a potent mitogen, proHB-EGF is also biologically active in two ways, one as a juxtacrine growth factor (15), and the other as a diphtheria toxin receptor (DTR) (16,17). It has also been shown that several growth factors and lymphokines are synthesized as membrane-anchored proteins, including the EGF family of growth factors, tumor necrosis factor-␣, colony-stimulating factor-1, and c-Kit ligands 1 and 2 (18). These transmembrane forms are biologically active. For example, the transforming growth factor-␣ precursor in co-culture stimulates EGFR phosphorylation, mitogenesis, and Ca ϩϩ uptake (19 -22). Transmembrane c-Kit ligand is required for the development of melanocytes, germ cells, and hematopoietic stem cells. Soluble c-Kit ligand cannot substitute for the transmembrane form in vivo (23)(24)(25).
An important feature of proHB-EGF is the formation of a complex with another transmembrane protein known as CD9 (17,26) and/or heparan sulfate proteoglycan (8) to express the maximal activities of both the juxtacrine growth factor and diphtheria toxin receptor (15,17,27). It has also been reported that proHB-EGF and CD9 form a complex with integrin ␣ 3 ␤ 1 on the cell surface (28); thus, integrin may affect the juxtacrine activity of proHB-EGF. Moreover, the carboxyl-terminal processing of proHB-EGF, which yields the soluble form, is regulated through a protein kinase C-induced mechanism, suggesting that an unidentified processing system is involved in the regulation of the juxtacrine growth factor activity (14). Therefore, the juxtacrine mechanism is a complex process and the mechanism is different from that of in the case of the soluble HB-EGF.
Using a co-culture system of donor cells expressing proHB-EGF in contact with acceptor cells expressing EGFR, we demonstrate here that amino-terminal processing of proHB-EGF is a requisite for full stimulation of cell growth in a juxtacrine manner. We also present the evidence that the amino-terminal processing does not affect the full expression of the paracrine growth factor activity of HB-EGF.

Materials
Sulfo-NHS (N-hydroxysuccinimide)-biotin was purchased from Pierce. Rabbit anti-human HB-EGF antibody H1 was raised against a synthetic peptide corresponding to amino acids 185-208 of the HB-EGF precursor, which are in the cytoplasmic domain, as described previously (29). An enhanced chemiluminescence (ECL) kit was purchased from Amersham (Buckinghamshire, United Kingdom). Recombinant human HB-EGF, produced in an Escherichia coli system (12), was a kind gift from Dr. Judith A. Abraham (Scios Inc., Mountain View, CA).

Cell Surface Biotinylation, Immunoprecipitation, and Detection of ProHB-EGF
CHO cells and transfectants were seeded in 10-cm dishes at a density of 1 ϫ 10 6 cells/dish and then incubated for 12 h. The cells were washed three times with ice-cold Hank's balanced salt solution, and then biotinylated with 0.1 mg/ml sulfo-NHS-biotin in 50 mM HEPES, pH 7.5, and 0.15 M NaCl for 15 min on ice. Excess reagent was quenched and removed by washing with ice-cold Dulbecco's modified Eagle's medium, 10% FCS. The cells were lysed with a lysis buffer (1% Triton X-100, 3 mM EDTA, 1 mM (p-amidinophenyl)methanesulfonyl fluoride HCl, 1 g/ml aprotinin, 10 mM antipain, 5 mM 3,4-dichloroisocoumarin, and 0.4 M NaCl in 20 mM HEPES, pH 7.2). After centrifugation at 15,000 rpm for 15 min, the supernatants were incubated with 10 g of rabbit anti-HB-EGF antibody H1 for 2 h at 4°C, followed by incubation with 10 l of protein A-Trisacryl (50% suspension) (Pierce) for 2 h at 4°C. The samples were dissolved in the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and then fractionated by 15% SDS-PAGE (33). Proteins in the gels were transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) in 150 mM CAPS buffer, pH 11, containing 20% methanol at 180 mA for 3 h. The nitrocellulose membranes were blocked with 5% skim milk in PBS (137 mM NaCl, 0.67 mM KCl, 8 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 ) overnight at 4°C. The membranes were then incubated for 30 min at room temperature with avidin-conjugated horseradish peroxidase (HRP) (Vector Laboratories Inc., Burlingame, CA). After being washed five times at intervals of 10 min with 0.05% Tween 20 in PBS, the membranes were treated with enhanced chemiluminescence (ECL) Western blotting detection reagents for 1 min (Amersham) at room temperature. The membranes were exposed to Kodak scientific imaging films (Eastman Kodak Co.). The densities of bands on the exposed films were determined with a Scanning Imager (Molecular Dynamics).

Northern Blot Hybridization
Total RNA was prepared from the cells according to the method of Chomczynski and Sacchi (34). RNA was electrophoresed on 1% agarose gel and was transferred onto a Zeta-probe membrane (Bio-Rad) by capillary action (35). The membrane filter was hybridized with 32 P-labeled monkey CD9 cDNA (36), a 0.6-kilobase pair XbaI-HindIII fragment at 42°C in hybridization buffer (35). The filter was washed with 2 ϫ standard saline citrate at 55°C, and exposed to a Fuji Film imaging plate in the BAS2000 bioimage analyzer (Fuji Photo Film) overnight.

Expression of Soluble HB-EGF in a Baculovirus-Insect Cell System
Expression of soluble HB-EGF in a baculovirus-insect cell system was carried out essentially by the method described previously (37).

Vectors
To express soluble forms of h⅐HB-EGFs and m⅐HB-EGFs, a stop codon was introduced at Pro 149 of pRc/h⅐HB-EGF and pRc/m⅐HB-EGF with a Mutant-K kit (Takara Shuzo, Ltd., Kyoto, Japan). The BamHI-XbaI fragment of mutated pRc/h⅐HB-EGF and the KpnI fragment of mutated pRc/m⅐HB-EGF were ligated into a baculovirus transfer vector pVL1393 (Invitrogen). The construction of mutant plasmids was confirmed by sequencing.

Expression of Recombinant Proteins
In expression experiments, Sf21 cells were infected with the recombinant viruses at a multiplicity of infection of 1-2. CM was harvested 72-96 h post-infection and used for HB-EGF purification.

Purification of Recombinant Soluble HB-EGF
Secreted h⅐HB-EGF produced by insect cells infected with recombinant baculovirus was purified from the insect cell CM by the method described previously with modifications (3). The insect cell CM (0.5 liters) was applied to a AF-Heparin Toyopearl 650 M column (2.5 ϫ 4 cm; Toso, Osaka, Japan) equilibrated with 0.5 M NaCl and 0.01 M Tris-HCl, pH 7.2. After extensive washing with the equilibration buffer, the bound proteins were eluted batchwise with 1.2 M NaCl and 0.01 M Tris-HCl, pH 7.2. The eluant was diluted 1:5 with 0.01 M Tris-HCl, pH 7.2, and then applied to a TSK-heparin 5PW column (8 ϫ 75 mm; Toso) equilibrated with 0.02 M NaCl and 0.01 M Tris-HCl, pH 7.2, using an FPLC system (Pharmacia). The column was washed with the equilibration buffer, and the bound proteins were eluted with a 40-ml linear gradient of 0.2-2 M NaCl in 0.01 M Tris-HCl, pH 7.2. The bio-active fractions were collected and applied to a C 4 reversed phase column (4.6 ϫ 250 mm; Vydac, Hesperia, CA) equilibrated with 5% acetonitrile in 0.05% trifluoroacetic acid, in a Shimadzu LC-10A HPLC system. The column was washed extensively with the equilibration buffer, and bound protein fractions were then eluted with a 60-ml gradient of 25-60% acetonitorile in 0.05% trifluoroacetic acid at the flow rate of 1 ml/min.

Amino Acid Composition and Amino-terminal Sequence Analyses of Recombinant Human HB-EGF
The purified h⅐HB-EGF (approximately 10 pmol) was applied to a Hitachi L-8500 amino acid analyzer (Hitachi, Tokyo, Japan) for amino acid composition analysis, and an Applied Biosystems model 492 microsequence analyzer together with an on-line Applied Biosystems model 610A phenylthiohydantoin-derivative analyzer (Perkin Elmer) for amino-terminal sequence analysis.

Growth Factor Assay
Soluble Growth Factor Activity Measurements-EP170.7 cells were washed with RPMI 1640 medium supplemented with 10% FCS, penicillin (100 units/ml), and streptomycin sulfate (100 g/ml). The cells (2 ϫ 10 4 ) were plated on 96-well plates in a total volume of 200 l. Appropriate volumes of samples were added to each well, and the EP170.7 cells were then incubated for 36 h. Ten l of [ 3 H]thymidine solution (1 Ci/10 l PBS, ICN Biomedicals Inc., Costa Mesa, CA) were added and after 4 h of incubation, the incorporation of [ 3 H]thymidine into DNA was measured using a 1205 Betaplate system (Pharmacia Biotech Inc.). Soluble recombinant HB-EGF (HB-EGF 75 or Arg 73 ⅐HB-EGF) (12) was used as a standard.
Juxtacrine Growth Factor Activity Measurements-The juxtacrine growth factor assay was carried out as described previously (15). Briefly, CHO transfectants (1 ϫ 10 5 cells/well) were plated in Ham's F-12, 10% FCS (500 l/well) in 24-well plates, and then incubated for 12 h prior to washing and fixation. The cells were washed twice with Ham's F-12, 10% FCS, 2 M NaCl to remove soluble HB-EGF trapped by cell surface heparan sulfate proteoglycan (38), and then fixed with 5% buffered formalin for 5 min. The formalin-fixed cells were washed twice with RPMI 1640, 10% FCS, and then EP170.

Juxtacrine Growth Factor Activities of Human and Mouse
HB-EGFs-It has been reported that cell surface proHB-EGF is able to stimulate adjacent cell growth through cell to cell contact (15). Since CHO cells did not show juxtacrine growth factor activity in our assay system, stable transfectants of CHO cells with h⅐HB-EGF or m⅐HB-EGF cDNA were cloned and subjected to the juxtacrine growth factor assay. One of the stable transfectants with h⅐HB-EGF cDNA, CHO h⅐proHB-EGF cells, showed significantly high juxtacrine growth factor activity as compared with that of a mock transfectant (Fig. 1A). In contrast, one of the cloned stable transfectants with m⅐HB-EGF cDNA, CHO m⅐proHB-EGF cells, showed extremely low juxtacrine growth factor activity (Fig. 1A). The expression levels of h⅐ and m⅐proHB-EGFs on both transfectants were estimated by the combination of biotinylation and immunoprecipitation methods. Although, as shown in Fig. 1B, the proHB-EGF protein levels were almost equal to each other, m⅐proHB-EGF appeared on an SDS gel as 22-27-kDa heterogeneous bands, which were much higher than the 19 -22-kDa heterogeneous bands of h⅐proHB-EGF. The same results were obtained when mouse L929 cells were used as host cells. 2 Endogenous CD9 Expression Is Enough for the Juxtacrine Growth Factor Activity in CHO Cells-Modulation of HB-EGF juxtacrine growth factor activity by CD9 has been reported (15). The difference of CD9 expression level in the two transfectants described in Fig. 1 might affect their juxtacrine growth factor activities. To investigate the implication of the difference between their CD9 expression levels, first CD9 mRNA expression was examined by Northern blot analysis, and second CD9 cDNA was transiently introduced into CHO h⅐proHB-EGF and CHO m⅐proHB-EGF . The endogenous CD9 mRNA was detected almost equally by Northern blot analysis ( Fig. 2A). Increased CD9 expression had no effect on the expression of their juxtacrine growth factor activities (Fig. 2B). These results suggest that the endogenous CD9 mRNA would be expressed enough as a co-factor for the juxtacrine growth factor. Based on these results, their molecular species might reflect their juxtacrine growth factor activities. Therefore, we studied proteolytic regulation of the juxtacrine growth factor activity using human/ mouse chimeric proHB-EGFs.
Processing of Human/Mouse Chimeric ProHB-EGFs-Soluble HB-EGF originally purified from U-937 CM showed molecular mass heterogeneity due to amino-terminal truncation. Since h⅐proHB-EGF has at least five potential cleavage sites (3), it is speculated that the heterogeneity of h⅐ and m⅐proHB-EGFs and their molecular mass difference would be due to amino-terminal truncation. The amino-terminal 67 amino acid sequences of h⅐ and m⅐proHB-EGFs were then replaced with each other to produce human/mouse chimeric proteins. Furthermore, a human epitope sequence for the #H1 antibody was conserved in the chimeras to facilitate quantitative analyses of the expressed proHB-EGF and its chimeras on CHO cells (Fig.  3A). A wild h⅐HB-EGF and three kinds of human/mouse chimeric cDNAs were transiently transfected into CHO cells, and the expressed proteins were analyzed by cell surface biotinylation and immunoprecipitation with the #H1 antibody. h⅐proHB-EGF migrated on an SDS gel as 19 -27-kDa heterogeneous bands, while m⅐proHB-EGF-H1 gave mainly 27-kDa band (Fig.  3B). Two other chimeras, h⅐(1-68)-H1 and h⅐(68 -208), migrated differently. Their migration patterns were almost the  ). B, immunoprecipitation of human and mouse proHB-EGFs. Cell surface proteins were biotinylated and immunoprecipitated with anti-HB-EGF antibody H1. The immunoreactive proteins were fractionated by SDS-PAGE (15% polyacrylamide), and after transfer to a nitrocellulose membrane, the biotinylated proteins were probed with avidin-HRP and chemiluminated with an ECL kit.

FIG. 2. CD9 mRNA expression in CHO m⅐proHB-EGF and CHO h⅐proHB-EGF cells.
A, Northern blot analysis of CD9 mRNA in CHO m⅐proHB-EGF and CHO h⅐proHB-EGF cells. Total RNA extracted from each cell was electrophoresed. Hybridization was performed with monkey CD9 cDNA according to the method described under "Experimental Procedures." B, effects of transient expression of CD9 on the juxtacrine growth factor activities of CHO m⅐proHB-EGF and CHO h⅐proHB-EGF cells. Mock or CD9 cDNA was transiently transfected into CHO m⅐proHB-EGF and CHO h⅐proHB-EGF cells. Their juxtacrine growth factor activities were measured according to the method described under "Experimental Procedures." The juxtacrine growth factor activity of CHO h⅐proHB-EGF was increased by the additional expression of h⅐proHB-EGF, suggesting that the proHB-EGF protein level produced by CHO h⅐proHB-EGF cells is appropriate for the estimation of CD9 as a juxtacrine up-regulator. same as those of h⅐proHB-EGF and m⅐proHB-EGF-H1, respectively (Fig. 3B). These results indicate that the molecular mass specificities of the human and mouse species are characterized by the amino-terminal 68-amino acid sequence, which would have different substrate specificities for some unidentified processing proteases.
Juxtacrine Growth Factor Activities of Human/Mouse Chimeric ProHB-EGFs-Since the juxtacrine growth factor activity of m⅐proHB-EGF was much lower than that of h⅐proHB-EGF, even though the m⅐proHB-EGF protein was produced in amounts equal to that of the h⅐proHB-EGF (Fig. 1, A and B), the human/mouse chimeric proHB-EGFs expressed on CHO cells were examined for juxtacrine growth factor activity. Each chimeric construct was transfected and stable clones with different expression levels were isolated to quantitatively analyze their juxtacrine growth activities. The juxtacrine growth factor activity and produced protein level of each clone were measured, as shown in Fig. 4 (A, B, D, and E). Quantitative comparison of their juxtacrine growth factor activities was carried out by plotting their activities against their protein expression levels. While m⅐proHB-EGF-H1 proteins weakly induced the growth of EP170.7 cells in a juxtacrine manner, wild type h⅐proHB-EGF showed significantly increasing juxtacrine growth promoting activity in parallel with the increase in the protein level on the cell surface (Fig. 4, B, C, E, and F), as also shown in Fig. 1A. Human/mouse chimeras, h⅐(68 -208), comprising h⅐proHB-EGF with the mouse amino-terminal sequence, showed approximately 5 times less specific activity in comparison with that of the wild type h⅐proHB-EGF at the initial linear phase (Fig. 4C). In contrast, human/mouse chimeras, h⅐(1-68)-H1, comprising m⅐proHB-EGF with the human amino-terminal and cytoplasmic sequence, showed approximately 8 times greater specific activity as compared with that of the wild type mouse proHB-EGF at the initial linear phase (Fig. 4F).
Mutual replacement of the amino-terminal portions of human and mouse proHB-EGFs caused drastic changes in their molecular species produced by CHO cells. h⅐(68 -208) chimeras were apparently less processed and composed of a major 27-kDa species, while the controls, h⅐proHB-EGFs, were composed of a major 19-kDa species (Fig. 4A). Furthermore, h⅐(1-68) chimeras were well processed and composed of a major 19-kDa species, while the controls, m⅐proHB-EGF-H1 proteins, were composed of a major 27-kDa species (Fig. 4D). Inhibition and acceleration of the processing of h⅐(68 -208) and h⅐(1-68)-H1 proteins were well correlated with up-and down-regulation of their juxtacrine growth factor activities, respectively (Fig. 4, A,  B, D, and E). Based on these results, the processing at the amino-terminal portion would be a requisite for full activity of the juxtacrine growth factor.
Paracrine Growth Factor Activity of Amino-terminally Extended HB-EGF-The paracrine growth factor activities of soluble h⅐HB-EGFs were estimated toward EP170.7 cells. The soluble h⅐HB-EGF produced by recombinant baculovirus-infected Sf21 cells existed in the CM as heterogeneous molecular forms (data not shown). The largest soluble form of h⅐HB-EGF was purified from the CM and migrated as a 19-kDa band on an SDS-PAGE gel. Amino-terminal sequencing analyses revealed that h⅐HB-EGF started at G 32 LAA.. of its primary translation form named Gly 32 ⅐h⅐HB-EGF (Fig. 5A). The specific activity of Gly 32 ⅐h⅐HB-EGF produced in the baculovirus system was identical to that of a 75-amino acid form of h⅐HB-EGF (Arg 73 ⅐h⅐HB-EGF) produced in an E. coli system with an ED 50 of 200 pM (12) (Fig. 5B). The Arg 73 ⅐h⅐HB-EGF has been reported to have the same specific activity as U-937-derived HB-EGF (12). These results indicate that the 41-amino acid extension at the aminoterminal end has no effect on the specific activity of h⅐HB-EGF as to the paracrine growth factor activity. Based on these results, amino-terminal truncation might have no effect on the paracrine growth factor activity of HB-EGF. DISCUSSION HB-EGF has been reported to associate with heparan sulfate proteoglycan and/or CD9 to express optimal mitogenic activity in juxtacrine and paracrine manners (8,15). In particular, on juxtacrine stimulation, complex formation of proHB-EGF with CD9 has been reported to be a requisite for the activation of EGFR on adjacent cells (15). This time, juxtacrine growth factor analysis of m⅐proHB-EGF provided us with a great opportunity to investigate the enzymatic activation mechanism of HB-EGF. Using a CHO cell system that was not affected by CD9 expression level, we demonstrated that amino-terminal truncation of proHB-EGF up-regulates its juxtacrine growth factor activity to a great extent, while it has no effect on its optimal paracrine growth factor activity.
Two kinds of human/mouse chimeric proHB-EGF with sub- stituted amino-terminal 67-amino acid sequences have been produced, and their protein production level and juxtacrine growth factor activities have been quantitatively analyzed. A human/mouse chimera with the mouse amino-terminal sequence appeared predominantly as a 27-kDa species on the CHO cell surface, and its juxtacrine activity was suppressed about 5-fold in comparison with that of the wild h⅐proHB-EGF. In contrast, a human/mouse chimera with the human aminoterminal sequence comprised a major 22-kDa species, and its juxtacrine growth factor activity was up-regulated about 8-fold as compared with that of the wild type m⅐proHB-EGF. These results lead us to the conclusion that the amino-terminal extension of HB-EGF suppresses its juxtacrine growth factor activity, and the amino-terminal processing is required for the optimal expression of its juxtacrine activity. However, it is intriguing that this conclusion is not applicable to the expression of its paracrine growth factor activity, because Gly 32 ⅐h⅐HB-EGF showed the same specific activity toward EP170.7 cells as Arg 73 ⅐h⅐HB-EGF. Although the precise molecular mechanism has not been elucidated yet, the amino-terminal extension sequence might interfere with the association with CD9 or the interaction of the complex with EGFR.
The existence of the R 57 DRKVR sequence in h⅐HB-EGF strongly indicates that the endoprotease, furin (39), could be involved in the amino-terminal processing (Fig. 6). This would . The immunoprecipitated protein was fractionated by SDS-PAGE, transferred to a nitrocellulose membrane, and then detected with avidin-HRP and an ECL kit. B and E, juxtacrine growth factor activity. The 12 cell lines mentioned in A and D were co-incubated with EP170.7 cells, and then their juxtacrine growth factor activities were measured as described under "Experimental Procedures." C and F, relationship between juxtacrine growth factor activities and protein production levels of proHB-EGFs. For each cell type, the intensities of the immunoreactive bands in the range of 19 -30 kDa in A and D were measured together with a Scanning Imager. The juxtacrine growth factor activities in B and E were plotted against the intensities of the proHB-EGFs in A and D. Symbols used are as follows:  be also supported by the facts that the 24-kDa species (Asp 63 ⅐h⅐HB-EGF shown in Fig. 5A) of the purified h⅐HB-EGF from U-937 cell CM was cleaved amino-terminally at its post R 57 DRKVR (3,37) and that h⅐HB-EGF was not processed in LoVo cells, which lack a functional furin, but was processed in LoVo/Fur 1 cells (40), which have an active furin. 2 In contrast, m⅐HB-EGF lacks the furin substrate sequence as shown in Fig.  6. The role of furin in the up-regulation of h⅐proHB-EGF juxtacrine growth factor activity must be further examined. The multiple amino-terminal truncation sites in h⅐HB-EGF also suggest the involvement of other proteases in its processing. m⅐HB-EGF would be cleaved amino-terminally at the multiple sites in vivo, too. Because proHB-EGF expressed in mouse uterus has been suggested to function as a juxtacrine growth factor (41,42).
We have also studied the effect of glycosylation on the juxtacrine growth factor activity of proHB-EGF. HB-EGF has two glycosylation sites, Thr 75 and Thr 85 . Since double mutations of these sites, Thr 75 3 Gly and Thr 85 3 Asn, had no effect on its juxtacrine activity, 3 the modification by glycosylation is not involved in the regulation of the juxtacrine activity.
The amino-terminal processing is the first report as posttranslational modification to affect the juxtacrine activity.
The physiological meaning of the amino-terminal processing of EGF superfamily members remains unknown. Not only HB-EGF but also transforming growth factor-␣, amphiregulin, ␤-cellulin, and neuregulins have multiple amino-terminal processing sites, which might suggest the physiological importance of their processing.

FIG. 6. Putative furin recognition site in human HB-EGF.
Alignment of the 53-80 amino acid sequences of the primary translation products of h⅐ and m⅐HB-EGFs. Arrowheads indicate amino-terminal processing sites in U-937 cells reported previously (3). Bold italic letters with underline denote the putative furin recognition site in h⅐HB-EGF, and bold letters without underline are the corresponding sequence in m⅐HB-EGF. The shaded regions of the h⅐ and m⅐HB-EGFs were used to make chimera HB-EGFs.