Cloning, characterization and developmental regulation of two members of a novel human gene family of neurite outgrowth-promoting proteins.

Growth Factors, 1991, Vol. 5, pp. 99-114 Reprints available directly from the publisher Photocopying permitted by license only

0 1991 Harwood Academic Publishers GmbH Printed in the United Kingdom

Cloning, Characterization and Developmental Regulation of Two Members of a Novel Human Gene Family of Neurite Outgrowth-Promoting Proteins PETER J. KRETSCHMER, JEANETTE L. FAIRHURST, MILDRED M. DECKER, CHRISTINE P. CHAN, YAKOV GLUZMAN, PETER BOHLEN and IMRE KOVESDI*

Growth Factors Downloaded from informahealthcare.com by UB Mainz on 10/28/14 For personal use only.

Molecular Biology Research Section, Medical Research Division, American Cyanamid Company, Lederle Laboratories, Pearl River, Nau York 10965

(Received February 22 1991, Accepted March 14 1991) This report describes the cloning, expression and characterization of two members of a novel human gene family of proteins, HBNF and MK, which exhibit neurite outgrowthpromoting activity. The HBNF cDNA gene codes for a 168-residue protein which is a precursor for a previously described brain-derived heparin-binding protein of 136 amino acids. The second human gene identified in this study, called MK, codes for a 143-residue protein (including a 22-amino acid signal sequence) which is 46% homologous with HBNF. Complementary DNA constructs coding for the mature HBNF and MK proteins were expressed in bacteria and purified by heparin affinity chromatography. These recombinant proteins exhibited neurite-outgrowth promoting activity, but lacked mitogenic activity. The HBNF gene is expressed in the brain of adult mice and rats, but only minimal expression of MK was observed in this tissue. Different patterns of developmental expression were observed in the embryonic mouse, with MK expression peaking in the brain between days El2 and E l 4 and diminishing to minimal levels in the adult, while expression of HBNF mRNA was observed to gradually increase during embryogenesis, reaching a maximal level at birth and maintaining this level into adulthood. Expression of these genes was also observed in the human embryonal carcinoma cell line, NT2/Dl. Retinoic acid induced the expression of HBNF and MK 6- and 11-fold, respectively, in this cell line. Our studies indicate that HBNF and MK are members of a new family of highly conserved, developmentally regulated genes that may play a role in nervous tissue development and/or maintenance. KEYWORDS: neurite outgrowth, human gene family, cDNA cloning, retinoic acid, NT2/Dl developmental regulation

embryonal carcinoma,

being grouped into gene families of similar sequence a n d function. For example, nerve growth factor (NGF), discovered over forty years a g o (Levi-Montalcini, 1987), has only recently been identified as a member of a family of neurotrophic factors (Leibrock e t al., 1989; Maisonpierre e t al., 1990). Similarly, acidic a n d basic fibroblast growth factors (aFGF a n d bFGF), both of which were identified fifteen years ago, are n o w recognized a s t w o members of a large family of growth stimulatory proteins (Baird a n d Bohlen, 1990). During studies on bovine brain aFGF a protein

INTRODUCTION Mammalian development is a complex process requiring the coordinated regulation of genetic expression mediated in part by a vast array of intercellular signalling factors. O n e such g r o u p of intercellular signals a r e the growth factors, proteins that have been implicated in a broad array of biological processes. As more of these factors a r e identified a n d characterized, they a r e “To whom correspondence should be addressed

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was noted which copurified throughout early purification steps and heparin affinity chromatography but which was separated from aFGF by high resolution chromatography (Bohlen et al., 1988; Bohlen and Gautschi-Sova, 1989). These initial studies suggested that the protein possessed mitogenic activity for endothelial cells and thus was termed heparin-binding brain mitogen, HBBM. N-terminal sequence analysis revealed that this protein was previously unidentified (Bohlen and Gautschi-Sova, 1989). The protein was subsequently also isolated by others from bovine brain and uterus (Rauvala, 1989; Milner et al., 1989; Kuo et al., 1990) and most of its sequence determined (Bohlen et al., 1991; Kuo et al., 1990). Interestingly, studies by Rauvala (1989) showed that the protein promotes neurite outgrowth in primary cultures of fetal rat neurons. This activity was confirmed by others (Milner et al., 1989; Bohlen, 1991). The mitogenic properties of the protein are the subject of controversy. Milner et al. (1989) and Li et al. (1990), using brain-derived and recombinant protein, respectively, found mitogenic activity for 3T3 fibroblasts but others were not able to demonstrate this activity (Kuo et al., 1990). Moreover, our own recent studies with highly purified protein showed a lack of mitogenic activity for endothelial cells and 3T3 fibroblasts while the same protein preparation displayed neurite-promoting activity (Bohlen et al., 1991). Various investigators have used different names for the protein, including p18 (Rauvala, 1989; Kuo et al., 1990), heparin-binding growth factor-8 (HBGF-8, Milner et al., 1989), pleiotrophin (PTN, Li et al., 1990). Based on its only confirmed activity, we have termed the protein HBNF (heparin-binding neurite-promoting factor). The N-terminal sequences of HBNFs from a variety of species (human, bovine, rat, chicken) were found to be identical or highly conserved (Huber et al., 1990). This suggests evolutionary pressure for sequence conservation and thus a potentially important biological function of HBNF. Recently, the high degree of interspecies sequence conservation has been confirmed by cDNA cloning of the HBNF gene from rat (Merenmies and Rauvala, 1990; Kovesdi et al., 1990; Li et al., 1990), bovine (Li et al., 1990) and human (this report; Li et al., 1990). Comparisons of the HBNF sequence with

sequences stored in the Protein Identification Resource/Genebank database revealed high sequence homology between HBNF and the protein encoded by the MK gene (Kovesdi et al., 1990; Bohlen et al., 1991). The MK gene is expressed during early stages of retinoic acidinduced differentiation of embryonic carcinoma cells, and during mid-gestation in mouse embryogenesis (Kadomatsu et al., 1988). Initially, the mouse MK gene was reported to code for a 90-residue protein but more detailed analysis indicated an encoded protein of 140 residues (Tomomura et al., 1990a). Mouse MK protein was found to be secreted by differentiating embryonal carcinoma cells and preliminary analysis of the secreted protein suggested heparin-binding properties and mitogenic activity for PC12 pheochromocytoma cells (Tomomura et al., 1990b). Based on in situ hybridization studies of MK and mRNA expression during mouse embryogenesis, Kadomatsu et al. (1990) suggested MK may play a fundamental role in the differentiation of a wide variety of cells, and that it may be involved in the generation of epithelial tissues and in the remodeling of mesoderm. Here we describe the cloning of the cDNAs of the human HBNF and MK genes and the expression of their biologically active proteins in E. coli. Further, we report on the developmental regulation of these genes in animals and in the human embryonal carcinoma cell line, NT2/Dl.

METHODS cDNA Cloning and Sequencing Lambda g t l l cDNA libraries prepared from the brain stem and basal ganglia of a one-day-old infant (constructed by Kamholz et al., 1986) were purchased from the ATCC (catalog numbers 37432 and 37433). A third lambda g t l l cDNA library representing mRNA from the brain of a 21-week fetus was purchased from Clontech (catalog number HL 1065b). Libraries were plated at a density of 30-40,000 PFU/150mm Petri dish on a lawn of E . coli LE 392, and the resulting plaques immobilized on nitrocellulose filters (Schleicher and Schuell, BA85), and hybridized to labeled probe as described by Sambrook et al. (1989). Double stranded DNA probes were labeled with [U-'~P]dCTP by random prim-

HUMAN HBNF AND MK GENES


ing using Klenow fragment of DNA polymerase I as described (Feinberg and Vogelstein, 1984). Purified bacteriophage clones were isolated following a secondary screen, high titre lysates prepared and DNA extracted as described previously (Sambrook et al., 1989). EcoRI inserts were subcloned into the pBS(+) plasmid (Stratagene), and sequenced on both strands by the dideoxy chain termination method (Sanger et al., 1977) using Sequenase reagents (United States Biochemicals). Unless stated otherwise, standard molecular biology techniques are as described by Sambrook et al. (1989). Polymerase Chain Reactions and Cloning of Amplified Fragments DNA fragments were amplified with primers ending with desired restriction endonuclease sites for subsequent cloning of the amplified fragment, as follows: (1)5‘CAAGCTTGCAACTGGAAGAAGGAATTTGAA3’, (HirzdIII); ( 2 ) 5’GGAATTCGGTCTCCTGGCACTGGGCAGT3’,(EcoRI); (3) 5’AAGGCATATGGGGAAGAAAGAGAAAC CAGAA-3’, (NdeI); (4) 5’GATTGGATCCTCTAGATTAATCCAGCA TCTTCTCCTG-3’, (BUMHI); (5)5’AAGGCATATGAAAAAGAAAGATAAGGTG-3’ (NdeI); (6) 5‘GATTGGATCCTCTAGACTAGTCCTTTCCCTT CCCTTTC-3’, (BUMHI). Polymerase chain reactions were carried out for 30 cycles as described by Saiki et al. (1985). For cloning MK sequence from mouse genomic DNA, primers 1 and 2 were used as sense and antisense primers with annealing temperatures of 50°C. For construction of the HBNF expression plasmid (pETHH8), amplification was at 45°C using the clone HHC8 and primers 3 (sense) and 4 (antisense). For construction of the MK expression plasmid (pETMH2), amplification was performed at 45°C using an MK cDNA clone as template and primers 5 (sense) and 6

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(antisense). Amplified fragments were purified by 1%agarose gel electrophoresis, isolated from the gel using DEAE-cellulose (Schleicher and Schuell, NA 45), digested with restriction endonucleases, and ligated with the appropriately digested plasmid DNA. The amplified mouse genomic fragment was cloned into pBS(+) plasmid (Stratagene). The HBNF and MK fragments were ligated into a derivative of the expression vector PET-3a (Studier et al., 19901, modified by deletion of the 1400 bp SalI/PvuII fragment and insertion of an f l origin of replication into the EcoRI site (I. Kovesdi, unpublished results), and transformed into BL21 LusS ., (Studier et al., 1990). The inserts of the expression plasmids were sequenced to confirm fidelity of amplification and cloning. Expression and Isolation of Recombinant HBNF and MK Proteins Single colony isolates containing the expression plasmids were grown and induced with IPTG as described (Studier et al., 1990). Pellets from one ml cultures were resuspended in 100 pl of SDS loading buffer (Laemmli, 1970) and 2.5 p1 run on a 15% acrylamide SDS-PAGE gel. Gels were stained with Coomassie blue. Recombinant HBNF and MK proteins were purified from bacterial cultures by chromatography on heparinSepharose CL-6B (Pharmacia) in 10 mM Tris, pH 7.0 using a linear salt gradient from 0.6 to 2.0 M NaCl. Proteins eluting between 1.0 and 1.3 M NaCl were further purified by cation exchange chromatography on a Mono-S (Pharmacia) column using a gradient from 0 to 1 M NaCl in 50 mM sodium phosphate, pH 6.8. HighIy purified HBNF and MK proteins were eluted at 0.6 M NaCI. Neurite Outgrowth Assays Brains from 18-day fetal rats were removed under sterile conditions and dispersed to single cells in Dulbecco’s modified Eagle media (DMEM) containing 10% FCS using a sterile 5 ml syringe. The cell suspension was adjusted to 5x lo5 cells/ml and plated onto tissue culture dishes that were precoated with 50 pg/ml poly-L-lysine for 30 min a t room temperature (Rauvala and Pihlaskari, 1987). Cultures were incubated for 24 hr at 37°C in 10% COz, after which the media

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was changed to DMEM containing 1 mg/ml BSA, and HBNF or MK proteins were added at indicated concentrations. After a further one-day incubation, neurite outgrowth activity was determined by visual examination of cells for extended outgrowth/processes as compared to controls.

foxide (10 p1) was added, and cells were incubated for 9 days. Fresh medium and RA were added at days 4 and 8. Plates were washed once with phosphate buffered saline, and RNA extracted as described above.

RESULTS


RNA Isolation and Blot Analysis Total cellular RNA from animal tissues or NT2/D1 cells was isolated by the guanidinium isothiocyanate-cesium chloride method (Chirgwin et al., 1979). For each RNA sample 10 pg was loaded per well on a 1%formaldehyde agarose gel (Lizardi et al., 1975) and electrophoresed for 16 hrs at 1.5 volts/cm. The gel was blotted onto nylon membrane filter (Gelman, BioTrace PR), and RNA was bound to the filter by UV irradiation with a UV Stratalinker model 2400 (Stratagene) at 120,000 pjoules/cm2. RNA blots were hybridized at 42°C in a solution of 50% formamide, 5xDenhardt’s solution, 5xSSPE (1xSSPE is 0.18 M NaC1, 10 mM NaP04, pH 7.4, 1 mM EDTA), 5 mM sodium pyrophosphate, 0.5% SDS, 50 pg/ml sonicated salmon sperm DNA and 32P-labeledprobes prepared by random oligonucleotide priming (Feinberg and Vogelstein, 1984). The filters were washed at 65°C in lxSSC (0.15 M NaC1, 15 mM Na-citrate, pH 7.0), 0.2% SDS and exposed to X-ray films. Human fetal RNA was purchased from Clontech.

Cloning and Comparison of Human HBNF and MK Genes

Both genes were cloned utilizing DNA probes generated by polymerase chain reaction (PCR) amplification (Saiki et al., 1985). Previously, we described a partial clone of the rat HBNF gene which was obtained by PCR amplification of rat cDNA utilizing degenerate primers corresponding to the known bovine HBNF amino acid sequence (Kovesdi et al., 1990). This rat clone, which codes for a contiguous 89 residues of HBNF, was used as probe to isolate human HBNF genes from lambda g t l l cDNA libraries prepared from the brain stem and basal ganglia of a one day old child (Kamholz et al., 1986). The EcoRI inserts from four positive lambda clones (Fig. ZAf were subcloned into the pBS(+) plasmid (Stratagene) for subsequent analysis. These four clones had identical nucleotide sequence in all overlapping regions except for HHC7, which had a three nucleotide in-frame deletion resulting in the removal of an alanine at position 119 (Fig. 1B). The significance of this deletion is unknown. The four cDNA clones accounted for 1383 of the Growth and Retinoic Acid Induction of the estimated 1600 nucleotides of the human HBNF Human NT2/D1 Cells mRNA as judged by northern analysis (Fig. 6B). The human embryonal carcinoma cell line The cDNA codes for a protein product of 168 NT2/Dl was grown as described previously amino acids, of which the first 32 residues appear (Andrews, 1984). For retinoic acid (RA) induc- to be a hydrophobic signal peptide sequence tion, cells were grown and resuspended in (von Heijne, 1985, 1986). The amino acid DMEM medium containing 10% bovine calf sequence following the 32 residues of the signal serum (Hyclone Laboratories, Inc) at a density o$ peptide corresponds exactly to the previously 5x105 cells per 100mm dish. Varying concen- published N-terminal sequence of human HBNF trations of all-trans retinoic acid in dimethyl sul- (Huber et al., 1990). Thus, mature human HBNF FIGFRE 1. Nucleotide and amino acid sequence of the human HBNF gene. (A) Four overlapping partial cDNA clones encoding HBNF. Top line indicates the mRNA with black and hatched boxes representing the HBNF coding region and postulated 3‘ poly(A) tract, respectively. Restriction sites are (H) HindlII; (K) KpnI; (P) PstI; nt indicates nucleotide length of clones. (B) Combined nucleotide sequences of clones HHC7, 8, 10 and 12 with the deduced amino acid sequence. Boldfaced amino acids represent the predicted protein signal peptide sequence (see text), and the arrow indicates the start of the mature protein. The two peptide sequences utilized for the design of oligonucleotide probes used in cloning the partial rat HBNF gene (Kovesdi et al., 1990) are underlined. The three nucleotides missing in clone HHC7 are boxed. Nucleotides representing stop codons in the 5’ non-coding regions are underlined.


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4 1 1 ATG CAG GCT CAA CAG TAC CAG CAG CAG CGT CGA AAA TTT GCA GCT GCC TTC TTG M Q A Q Q Y Q Q Q R R K P A A A P L -32 5 3 1 GCA TTC ATT TTC ATA CTG GCA GCT GTG GAT ACT GCT GAA GCA GGG AAG AAA GAG A -14 F I P I L A A V D T A E A E K K E 5 8 5 AAA CCA GAA AAA AAA GTG AAG AAG TCT GAC TGT GGA GAA TGG CAG TGG AGT GTG P E K K V K K S D C G E W Q W S V 5 K

639 TGT GTG CCC ACC AGT GGA GAC TGT GGG CTG GGC ACA CGG GAG GGC ACT CGG ACT V P T S G D C G L G T R E G T R T 23 C 6 9 3 GGA GCT GAG TGC AAG CAA ACC ATG AAG ACC CAG AGA TGT AAG ATC CCC TGC AAC A E C K Q T M K T Q R C K I P C N 41 G 1 4 1 TGG AAG AAG CAA TTT GGC GCG GAG TGC AAA TAC CAG TTC CAG GCC TGG GGA GAA K K Q F G A E C K Y Q F Q A W G E 59 W 8 0 1 TGT GAC CTG AAC ACA GCC CTG AAG ACC AGA ACT GGA AGT CTG AAG CGA GCC CTG 77 C D L N T A L K T R T G S L K R A L 8 5 5 CAC AAT GCC GAA TGC CAG AAG ACT GTC ACC ATC TCC AAG CCC TGT GGC AAA CTG N A E C Q K T V T I S K P C G K L 95 H 9 0 9 ACC AAG CCC AAA CCT CAA GCA GAA TCT AAG AAG AAG AAA AAG GAA GGC AAG AAA 113 T K P K P Q M E S K K K K K E G K K 9 6 3 CAG GAG AAG ATG CTG GAT TAA E K M L D * 131 Q 981 1054 1124 1194 1264 1334

RAGATGTCACCTGTGGAACATAAAAAGGACATCAGCAAACATCAGCAAACAGGATCAGTTAACTATTGCATTTATATGTA CCGTAGGCTTTGTATTCATTATCTATAGCTAAGCTAAGTACACAATAAGC~C~CC~T~TGGGTTC TGCAGGTACATAGAAGTTGCCAGCTTTTCTTGCCATCCTCGCCATTCG~TTTCAGTTCTGTACATCTGC CTATATTCCTTGTGATAGTGCTTTGCTTTTTCATAGATAAGCTTCCTCCTTGCCTTTCGAAGCATCTTTT GGGCAAACTTCTTTCTCAGGCGCTTGATCTTCAGCTCTGCG~TTCCTTCGCTTTTTCTTAAGGGTTTC TGGCACAGCAGGAACCTCCTTCTTCTTCTCTTCTACACCCTCTATGTACC FIGURE 18

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AAG AAA GAT AAG GTG AAG AAG GGC GGC CCG GGG AGC GAG K K D K V K K G G P G S E

134 TGC GCT GAG TGG GCC TGG GGG CCC TGC ACC CCC AGC AGC AAG GAT TGC GGC GTG K D C G V P S S C T 1 5 C A E W A W G P 188 GGT TTC CGC GAG GGC ACC TGC GGG GCC CAG ACC CAG CGC ATC CGG TGC AGG GTG Q R I R C R V 3 3 G F R E G T C G A Q T


242 CCC TGC AAC TGG AAG AAG GAG TTT GGA GCC GAC TGC AAG TAC AAG TTT GAG AAC K Y K F E N C F G A D 5 1 P C N W K K E 296 TGG GGT GCG TGT GAT GGG GGC ACA GGC ACC AAA GTC CGC CAA GGC ACC CTG AAG L K 6 9 W G A C D G G T G T K V R Q G T 350 AAG GCG CGC TAC AAT GCT CAG TGC CAG GAG ACC ATC CGC GTC ACC AAG CCC TGC 8 7 K A R Y N A Q C Q E T I R V T K P C 404 ACC CCC AAG ACC AAA GCA AAG GCC AAA GCC AAG AAA GGG AAG GGA AAG GAC TAG 105T P K T K A K A K A K K G K G K D * 458 528 598 668 738

ACGCCAAGCCTGGATGCCAAGGAGCCCCTGGTGTCACATGGGGCCTGGCCACGCCCTCCCTCTCCCAGGC CCGAGATGTGACCCACCAGTGCCTTCTGTCTGCTCGTTAGCTTTAATCAATCATGCCCTGCCTTGTCCCT CTCACTCCCCAGCCCCACCCCTAAGTGCCCAAAGTGGGGAGGGACAAGGGATTCTGGGAAGCTTGAGCCT CCCCCAAAGCAATGTGAGTCCCAGAGCCCGCTTTTGTTCTTCCCCACAATTCCATTACTAAG~CACAT CAAATAAACTGACTTTTTCCCCCCAATWGCTCTTCTTTTTTAATAT-

FIGURE 2. Nucleotide and amino acid sequences of the human MK gene. Boldfaced amino acids represent the predicted protein signal peptide sequence, and the arrow represents the predicted N-terminus of the mature protein. The two peptide sequences corresponding to primers 1 and 2 (see Methods) used to amplify the mouse genomic DNA probe (see Results) and the two polyadenylation sequences near the 3’ end of the gene are underlined.

is a 136-residue protein with a calculated molecular weight of 15.3 kDa. It should be noted that HBNF appears to be an 18 kDa protein as judged by SDS-PAGE (Fig. 4), but this molecular weight is artifactually high, most likely because of aberrant migration of the highly basic protein. The same phenomenon has been observed with the mouse MK protein (Tomomura et al., 1990a). To clone the human MK cDNA, PCR was used to amplify mouse genomic DNA with sense and antisense primers corresponding to two regions of the mouse cDNA separated by approximately

150 nucleotides (Fig. 2). Cloning and sequencing of the resultant DNA fragment revealed a sequence corresponding to 150 nucleotides of the published mouse MK cDNA clone (Tomomura et al., 1990a; data not shown). This sequence was then used as a probe to screen two human lambda g t l l cDNA libraries, one prepared from the stem cells of a one-day infant (used above), and a second prepared from the brain of a 21week human fetus. The EcoRI inserts from positive clones were isolated, subcloned into pBS(+) and sequenced. The sequence of one of these

FIGURE 3. (A) Sequence homology between human HBNF and MK proteins. Vertical lines between the sequences indicate identical amino acids. Boxed amino acids indicate conserved cysteine residues. (B) Sequence homology between human and mouse MK proteins. Boldfaced amino acids indicate differences between the two proteins. Dashes indicate breaks used to achieve maximum sequence identity in both figures (A) and (B).


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clones, MKHC4, is shown in Fig. 2, and accounts for 790 nucleotides of the estimated 1100 nucleotides of mature human MK mRNA (data not shown). The nucleotide sequence was subsequently confirmed in additional shorter-length MK clones, which were found to contain differing overlapping fragments of the MKHC4 clone (data not shown). The sequence of the MK cDNA includes two polyadenylation signals and a polyA tail (Fig. 2). Therefore, approximately 200-300 nucleotides at the 5' end of the MK mRNA are still unidentified. The MKHC4 clone has an open reading frame with a coding region beginning at nucleotide 22 and defining a 143 residue protein. The N-terminal sequence is highly hydrophobic and has the characteristics of a signal peptide (von Heijne, 1985). On the basis of the criteria for signal peptide structures proposed by von Heijne (1985, 1986), and comparisons with mouse MK and human HBNF sequences we assume that signal peptide cleavage occurs between amino acid residues 22 (Ala) and 23 (Lys) thus giving rise to a mature MK polypeptide of 121 residues in length (Fig. 2). Figure 3A shows the homology between the human HBNF and MK protein sequences. Of the 121 residues of the mature MK protein, 61 (50%) are identical to corresponding residues of the HBNF protein. Most significant, all 10 cysteine residues in both proteins are perfectly aligned, suggesting similar 3-dimensional structures and, perhaps, similar biological activities. The human

FIGURE 4. Bacterial Expression of human recombinant HBNF and MK proteins. Cell lysates were from bacterial cultures containing the expression plasmids pETHH8 (HBNF) or pETMH2 (MK). Lanes 1 and 2, lysates from uninduced and IPTG-induced cultures containing pETMH2. Lane 3, purified recombinant MK protein. Lanes 4 and 5, uninduced and induced cultures containing pETHH8. Lane 6, purified recombinant HBNF protein.

and mouse MK protein sequences are compared in Fig. 3B. The 140-residue mouse sequence can be aligned with the 143-residue human sequence such that 90% (126/140) of the mouse amino acids are identical to those of the human sequence. Biological Properties of HBNF and MK Proteins To provide a source of both mature proteins free of contaminating eukaryotic proteins, cDNA clones isolated above were used as templates for PCR amplification with primers designed to place a methionine codon immediately 5' of the N-terminal glycine (for HBNF) or lysine (for MK1 residues of the mature proteins. The amplified products were cloned into a modified form of the expression vector PET-3a (Studier et al., 1990), and the resulting plasmids, pETHH8 (for HBNF) or pETMH2 (for MK) were transformed into E . coli strain BL21 LysS. Protein extracts from isopropyl-P-D-thiogalactopyranoside (1PTG)induced pETHH8-containing bacteria revealed considerable quantities of protein migrating at approximately 18 kDa (Fig. 4, lane 51, which corresponds to that seen for native bovine HBNF (data not shown). Protein extracts of IPTGinduced pETMH2-containing bacteria showed a major protein band migrating at approximately 16.5 kDa (Fig. 4, lane 2). Uninduced cultures (lanes 4 and 1, for pETHH8- and pETMH2-containing bacteria, respectively) contained much



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FIGURE 5. Neurite outgrowth assays of purified recombinant HBNF and MK proteins. Purified proteins were assayed on 18day fetal rat neurons at concentrations indicated. (A) Neuronal cells with no added protein. (B) Bovine brain-derived HBNF protein (160 ng/ml). (C) Purified recombinant human HBNF protein (150 ng/ml). (D) Purified recombinant human MK protein (150 ng/ml).

less of these proteins as judged by SDS-PAGE aortic endothelial cells and NIH 3T3 fibroblasts band intensities. Recombinant HBNF and MK as described previously (Bohlen et al., 1991). The proteins were purified from IPTG-induced bac- recombinant MK protein exhibited no mitogenic terial cultures by heparin affinity chromatogra- activity (data not shown). In contrast to findings phy (Fig. 4, lanes 6 and 3, respectively), and their by others (Li et al., 1990), recombinant HBNF N-terminal sequences and amino acid compo- protein lacked mitogenic activity (data not shown). This is consistent with more recent sitions confirmed (data not shown). Recombinant HBNF and MK proteins were observations in this laboratory (Bohlen et al., assayed for the ability to stimulate neurite out- 1991), and elsewhere (Kuo et al., 1990) that highly growth of 18-day fetal rat brain neurons. Both purified preparations of brain-derived HBNF are bacterially-derived proteins showed neurite out- devoid of mitogenic activity. growth-promoting activity similar to that of native bovine HBNF (Fig. 5). The recombinant Expression of HBNF and MK Genes proteins were also assayed along with bovine HBNF for mitogenic activity on adult bovine Tissue-specific expression of HBNF and MK


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FIGURE 6. Northern blot analysis of HBNF mRNA in tissues of the mouse and the human brain. (A) From each adult mouse tissue 20 p g of total RNA was applied per lane. (B) Comparison of 1Opg of total RNA from embryonic human and mouse brains.

genes was examined in the adult mouse. Northern hybridization analysis of total RNA from a variety of adult tissues indicated that only the brain expressed a 1.65 kb band corresponding to mouse HBNF mRNA (Fig. 6). MK expression was not detected in any of these adult tissues. For HBNF, this expression is consistent with the presence of HBNF protein in human, bovine, rat and chicken brain tissues (Huber et al., 1990; Rauvala, 1989). To more fully localize brain HBNF gene expression, RNA extracts from various regions of the brain of 2-month-old rats were examined. As shown in Fig. 7, HBNF mRNA was readily detected in all regions examined, indicating a generally broad expression of the HBNF gene throughout the brain. When the same blot was hybridized to the MK probe, MK mRNA was detected in two brain regions, the caudate nucleus and the brain stem. Based on the significantly longer exposure times needed to see these bands in adult RNA as compared to equivalent amounts of embryonic RNA, it would appear that MK RNA is expressed at minimal levels in the adult. This, together with the limited regional

specificity of expression, most probably explains why our initial experiments (Fig. 6) failed to detect MK gene expression in adult mouse brain. The time course of developmental expression of MK and HBNF was examined in the brains of embryonic rats. Low levels of HBNF mRNA were detected in early embryonic stages, increased to a maximal level at or just after birth, and remained at a relatively constant level into the adult (Fig. 8). In contrast, expression levels of the MK gene were low in early embryos and rapidly peaked between days El2 and E14, followed by an equally rapid fall until birth, when expression was barely detectable. As noted above, a low level of MK expression is maintained into adult life, at least in the brain. The embryonic brain expression results are in general agreement with the in situ hybridization studies of Kadomatsu et al. (1990). In these experiments lower levels of MK expression were detected at days E7 and E9, with a rise in intensity of MK expression in a limited number of tissues, including the brain, at days E l l and E13. By day E15, this intense expression had been reduced significantly such that MK expression was only detected in the kid-


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both genes followed a similar pattern (Fig. 9). Levels of mRNA expression remained at a steady background level with 0.01-0.05 pM RA, rapidly increased between 0.1 and 0.5 pM RA, and maintained this level at concentrations u p to and including 10 pM RA. When RNA hybridization signals were normalized to a control p-actin probe, the maximum increases were calculated to be 6-fold for HBNF and 11-fold for MK (Fig. 98). These results are comparable to those observed for MK during retinoic acid induction of the mouse EC cell line, HM-1 (Kadomatsu et al., 1988). In this cell line, MK gene expression was induced 8-10-fold above background (Tomomura et al., 1990a).

DISCUSSION

FIGURE 7. Gene expression of HBNF and MK in rat brain. RNA extracted from various brain regions of 2-month-old rats was subjected to northern analysis (10 pg of total RNA/lane). The resulting blot was hybridized consecutively to probes for HBNF, MK, and B-actin.

ney. We were unable to detect MK mRNA expression in kidney tissue. Retinoic Acid Induction of HBNF and MK Gene Expression in NT2 Human Teratocarcinoma Cells The human embryonal carcinoma (EC) cell line NT2/D1 can be induced to differentiate at concentrations of retinoic acid (RA) varying from 0.01 to 10pM, with the proportion of differentiating EC cells ranging from 50% at 0.01 pM RA (Simeone et al., 1990) to greater than 99% at 1 and 10 pM RA (Andrews, 1984). Therefore, we studied the expression of the human HBNF and MK genes during differentiation of NT2/D1 cells at concentrations of RA ranging from 0.01 to 10 pM. After nine days exposure to RA, total RNA was extracted from cells and probed for gene expression by northern analysis. Expression of

In the current study we have isolated and characterized two human genes which define a new family of evolutionary conserved proteins with neurite outgrowth-promoting properties. The HBNF protein appears to be extraordinarily conserved throughout evolution. For example, bovine (Bohlen et al., 1990; Li et al., 1990) and human HBNF differ only in the conservative replacement of two out of 136 amino acids, while the bovine and rat (Kovesdi et al., 1990; Merenmies and Rauvala, 1990; Li et al., 1990) HBNF sequences differ by one amino acid. Thus, all genes and proteins so far characterized in this family have strictly maintained two features, namely, location of cysteine residues and high basic amino acid content. Human HBNF and MK, bovine HBNF, rat HBNF, and mouse MK proteins all have ten cysteine residues at invariant positions, strongly suggesting that these proteins have very similar three dimensional structure and, potentially, similar function. The biological activities of the MK and HBNF proteins are still poorly understood. While the neurite outgrowth-promoting activity of HBNF is well established, there is still controversy as to whether this molecule also possesses mitogenic activity. In support of our previous studies with native HBNF (Bohlen et al., 19911 and those of others (Kuo et al., 1990), we did not detect any mitogenic activity of recombinant HBNF. However, another group reported mitogenic activity of both the native (Milner et al., 1989) and recombinant proteins (Li et al., 1990). With respect to


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FIGLIFE 8. HBNF and MK gene expression in rat development. RNA was extracted from whole embryos (for days E8, ElO), embryonic brains (days E12-E20) and brains of twoday-old (P2) and adult rats (A). Total RNA (10pg/lane) was subjected to northern analysis and consecutively hybridized with HBNF, MK and p-actin probes.

MK less data is available. Conditioned medium from MK-transfected L cells secreting MK protein was reported to be mitogenic for PC12 cells (Tomomura et al., 1990b) and the current study provides the first evidence that MK, like HBNF, promotes the outgrowth of neurites. However, we did not find any mitogenic activity of recombinant MK for endothelial cells and 3T3 fibroblasts. It was somewhat surprising that recombinant HBNF and MK expressed in E. coli possessed bioIogical activity. Since both proteins contain many cysteines (most of which probably exist in a state of disulfide bonds) it was considered likely that incorrect protein folding during biosynthesis in

E . coli would occur, thus rendering the recombinant proteins inactive. In experiments not reported in this study, we did indeed find evidence suggestive of improper folding of both HBNF and MK. First, heparin affinity chromatography retained only a relatively small proportion of the proteins represented by the gel bands corresponding to HBNF and MK expressed proteins in E . coli extracts (Fig. 4, lanes 5 and 2, respectively). Furthermore, the fraction of the proteins that was retained by heparin affinity chromatography was found to yield multiple peaks on Mono-S cation exchange chromatography, all of which were related to HBNF or MK as indicated by N-terminal sequencing. Finally,

FIGURE 9. Expression of HBNF and MK genes in cultured NT2/Dl cells after 9 days of retinoic acid treatment. (A) For each RA concentration, 10 pg of total RNA was used in northern analysis. The resulting blot was consecutively hybridized with HBNF, MK and p-actin probes. (B) Hybridization signals obtained in Figure 9A for HBNF (black) and MK (hatched) were measured by densitometry and normalized to the p-actin signals.



Actin

HBNF

MK

B

RA (pM)

FIGURE 9

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KRETSCHMER et al.

when these heterogeneous protein preparations were run on Mono-S chromatography in the presence of a reducing agent, apparent peak heterogeneity disappeared and a single protein peak was observed. This strongly suggests that disulfide scrambling occurred during protein folding (probably not during purification because similar heterogeneity was never observed with brainderived HBNF), leading to multiple forms of proteins. The fact that preparations of recombinant HBNF and MK displayed neurite outgrowth activity suggests that at least some of the correctly folded proteins were synthesized in the bacteria, or that some of the improperly folded but heparin-binding molecular species retained significant activity. The developmental regulation of HBNF gene expression is temporally and spatially different from MK. HBNF gene expression was detected in the developing rat between embryonic days El0 and E l 2 with a gradual increase in the brain until birth, after which it remains approximately constant until at least 2 months after birth (Fig. 8.). Comparable results have beeen reported in the rat by Merenmies and Rauvala (1990)who also detected minimal early postnatal expression in the kidney and heart, with disappearance of expression by day 28. Similarly, Li et al. (1990) reported that HBNF was expressed just prior to birth of rats and decreased (to still significant levels) by postnatal day 22 in the rat brain. They also detected HBNF expression at birth primarily in the gut and striated muscle. In general agreement with our results, these studies detected minimal HBNF mRNA in day 13 rat embryos. Thus, the initial studies on developmental regulation of HBNF gene expression support a role for this protein in late embryonic to adult development. The site of this expression is primarily in the brain although lower levels of developmentally regulated expression may be found in other tissues (Milner et al., 1990; Li et al., 1990). In contrast to HBNF, MK gene expression appears much more confined to a specific stage in embryonic development. MK gene expression was dramatically increased at days E12-El4, after which it decreased to significantly lower levels at birth (Fig. 8). A similar temporal control of MK gene expression was found during in situ hybridization studies of mouse embryogenesis (Kadomatsu et al., 1990).In this analysis, limited

expression was observed in a variety of tissues at days E7 and E9. Expression was more prominent in many tissues, including the brain, at days Ell-E13, and then decreased rapidly such that by day El5 only the kidney expressed significant levels of MK mRNA. Reduced levels persisted into adulthood. As discussed above, we have not observed MK expression in the kidneys of 2month-old mice. However, in adult rats MK expression was found primarily in two regions of the brain, the caudate nucleus and the brain stem (Fig. 7). In general, results to date are consistent with the notion that MK protein plays a temporally restricted role in embryonic development. Analogous to HBNF, this expression occurs primarily in the brain, but may be detected in other tissues. An important tool in understanding the developmental regulation of human HBNF and MK gene expression would be a model system for human differentiation. One cell line with the potential to fill such a role is the clonally derived, pluripotent human embryonal carcinoma cell line, NT2/Dl (Andrews, 1984). This EC line, a derivative of the teratocarcinoma Tera-2 (Fogh and Trempe, 1975), undergoes differentiation into several cell types, including neurons, when retinoic acid is added to the culture medium (Andrews et al., 1984;Lee and Andrews, 1986).In particular, there is evidence that NT2/D1 cells induced with retinoic acid provide a model system for studies of human neuronal differentiation (Lee and Andrews, 1986).Our finding that HBNF and MK genes can be induced 6- and 11fold, respectively, by retinoic acid induced differentiation of NT2/Dl cells may provide an important model cell system for regulation of these genes, and may be valuable in defining the role of these proteins in neuronal cell development. In summary, HBNF and MK are two members of a new family of highly conserved proteins with neurite outgrowth-promoting activity. The differential regulation of HBNF and MK gene expression during development suggests that these proteins have functions related to tissue development. Furthermore, their prominent expression in the brain may indicate that these proteins are involved specifically in the development and/or maintenance of neural tissues. Finally, their presence in the brain and their neurite outgrowth-promoting capabilities raise


the possibility that these proteins are additional candidates for the growing list of neurotrophic factors (Baird and Bohlen, 1990; Leibrock et al., 1989; Lin et al., 1990; Maisonpierre et al., 1990; Rosenthal et al., 1990; Scott et al., 1983).

ACKNOWLEDGEMENTS We thank Douglas C. Armellino for technical h e l p w i t h the mitogenic activity assays.


SEQUENCE ACCESSION NUMBERS The human HBNF sequence data will appear in GenBank nucleotide sequence data bank under accession number M57399 and human MK in the EMBL data bank under accession number X55110.

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