The Kallmann syndrome gene product expressed in COS cells is cleaved on the cell surface to yield a diffusible component
The Kallmann syndrome gene product expressed in COS cells is cleaved on the cell surface to yield a diffusible componentElena I. Rugarli1,*, Cristina Ghezzi1, Valentina Valsecchi1 and Andrea Ballabio1,2
1Telethon Institute of Genetics and Medicine, San Raffaele Biomedical Science Park, via Olgettina 58, 20132 Milano, Italy and 2Dipartimento di Biologia Molecolare, University of Siena, Siena, Italy
Received February 28, 1996;Revised and Accepted March 29, 1996
Kallmann syndrome is characterized by hypogonadotropic hypogonadism and anosmia and caused by a defect of migration and targeting of gonadotropin- releasing hormone-secreting neurons and olfactory axons during embryonic development. We previously cloned the gene responsible for the X-linked form of the disease encoding a 680 amino acid protein, KAL, which displays the unusual combination of a protease inhibitor domain with fibronectin type III repeats. Previous expression studies by northern blot and RNA in situ hybridization in human and chick indicated that the gene is expressed at very low levels in the olfactory bulb during development. Therefore, low abundance of the protein has hampered a detailed biochemical characterization. By overexpressing both the human and chick KAL cDNAs in eukaryotic cells, we now provide evidence that KAL is a glycosylated peripheral membrane protein with an apparent molecular weight of approximately 100 kDa. We show that this 100 kDa protein is proteolytically processed on the cell membrane to yield a 45 kDa diffusible component, which is detectable with an antisera against the C-terminal part of the protein and binds tightly to cell surfaces. These data provide a first step toward understanding KAL function in neuronal interactions and neurite extension in the olfactory bulb and suggest that KAL might be a diffusible chemoattractant molecule for olfactory axons.
Kallmann syndrome is caused by a specific developmental defect within the olfactory system (1 ,2 ). Targeting of olfactory axons to the olfactory bulb and migration of neurons secreting the gonadotropin-releasing hormone (GnRH) are impaired. Both olfactory and GnRH-secreting neurons originate in the olfactory placode, which will later develop into the olfactory epithelium (3 ,4 ). During normal development, olfactory neurons send their axons to the olfactory bulb, while GnRH neurons migrate along the pathway of the olfactory nerve and through the olfactory bulb until they reach the hypothalamus. In Kallmann syndrome these processes are incomplete: migration of both olfactory axons and GnRH neurons is arrested within the meninges above the cribriform plate (5 ). As a result, Kallmann syndrome patients suffer from an inability to smell (anosmia) and hypogonadotropic hypogonadism due to a deficiency of GnRH (for a review see ref. 6 ).
We and others have previously cloned the gene responsible for the X-linked form of Kallmann syndrome (KAL) (7 -10 ) and its chick homologue (KALc) (11 ,12 ), which are 77% identical at the amino acid level. Both KAL and KALc have been found to be developmentally expressed in the olfactory bulb (11 -14 ), which is the central target of olfactory axons and the path through which GnRH-secreting neurons migrate. On this basis, it has been proposed that KAL might play a part in migration and targeting of olfactory axons and GnRH neurons within the olfactory bulb and not in the initial migration from the olfactory placode to the forebrain. Expression in the olfactory bulb is target-autonomous, i.e. independent from proper innervation by olfactory axons (13 ). KAL expression was also found in the human and chick cerebellum, retina and developing kidney, in the human developing cerebral cortex and thalamus and in the chick developing limbs (11 -14 ).
KAL encodes a 680 amino acid predicted protein without any obvious transmembrane domain or site of linkage to the membrane. The presence of a signal peptide suggests that the protein may be secreted. KAL shares significant homologies with molecules known to play a part in neural development. The N-terminus of the molecule displays a `four-disulfide-core domain', a protein motif characterized by cysteine residues with a conserved spacing and found in protease inhibitors of the serine kind (15 ). There is emerging evidence that nerve growth cones express proteases on their surface to facilitate migration through the extracellular matrix (16 -18 ). An attractive idea is that KAL is a secreted molecule which functions by altering the matrix-digesting activity of nerve growth cones.
The protease inhibitor domain is followed by four fibronectin type III (FNIII) repeats, a very common protein module described first in fibronectin (19 ) and then in several other extracellular matrix molecules as well as in cell-adhesion molecules, protein kinases and tyrosine-phosphatases (20 ,21 ). Several of these molecules have been implicated in the processes of neuronal migration and axonal targeting (22 -25 ). The FNIII repeats present in KAL are more similar to those found in the cellular adhesion molecules L1 (26 ), TAG-1 (27 ), F3 (28 ) and contactin (29 ). L1 has recently been proven to be involved in X-linked hydrocephalus, another human neuronal migration defect (30 ,31 ). TAG-1, F3 and contactin mediate neurite outgrowth and reciprocal axonal interactions through the complex display of both adhesive and anti-adhesive properties.
The neuronal migration defect in Kallmann syndrome and the developmental expression of the gene in the olfactory bulb are two factors that raise the question of whether or not KAL is a diffusible guidance molecule which could act as a chemoattractant for olfactory axons and GnRH neurons. To address this question, we analyzed biosynthesis and processing of KAL by expressing the protein in eukaryotic cells. We now present evidence that both KAL and KALc proteins are glycosylated and associated to the cell surface and, after proteolytic cleavage, they are secreted into the extracellular matrix.
Antisera were raised against the synthetic peptide NH2-SLVPTKKKRRKTTDG-COOH, corresponding to positions 340-354 of the human KAL protein (KL; Fig. 1 A). Ten of 15 amino acids of this peptide are conserved in the chick KALc protein. Polyclonal antibodies were also generated by immunizing rabbits against a recombinant GST-KALc fusion protein expressed in Escherichia coli, containing the last 219 amino acids of the KALc protein (ERB; Fig. 1 A). These comprise half of the third FNIII repeat and the entire fourth FNIII repeat. The level of homology within the fourth FNIII repeat between chick and human is close to 90% (Fig. 1 A).
Conditions for solubilization of KAL antigen from transfected COS cells
Extraction buffer
Recovery of KAL antigen in extraction buffer
6 M guanidine-HCl, 50 mM Tris-HCl pH 7.5
Yes
1 M NaCl, 50 mM Tris-HCl pH 7.5, 1% Nonidet P-40
Yes
0.1 M phosphate buffer pH 5.8, 1% Triton X-100, 1 mM EDTA
Yes
150 mM NaCl, 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM DTT, 0.1% SDS
No
150 mM NaCl, 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1% Nonidet P-40
No
6 M urea, 50 mM Tris-HCl pH 7.5
No
0.1 M phosphate buffer pH 6.8, 1% Triton X-114
No
To begin functional characterization of the protein, we transiently expressed both KAL and KALc cDNAs in COS 7 cells. We designed expression constructs to yield c-myc-tagged KAL or KALc proteins. To this end, we cloned the KAL and KALc coding regions with the c-myc epitope in frame at the C-terminus into a modified pMT21 expression vector (Fig. 1 B). Analysis of recombinant protein was initially performed by western blot. In transfected COS 7 cells, both ERB and KL antisera detected two major bands with an apparent molecular weight of 85 and 100 kDa. The same bands were detected in transfected COS 7 cells by the anti-c-myc monoclonal antibody, Mab 9E10 (32 ) and were not present in mock-transfected cells (Fig. 2 A). The KL antisera cross-reacted with both the KAL and KALc proteins, while the ERB antisera recognized the KALc protein with high affinity and the human KAL protein with much lower affinity (Fig. 2 A). These 85 and 100 kDa components of the KAL antigen are most likely peripheral membrane proteins, as they are released by the membranes after treatment with high salt, low pH and chaotropic agents such as 6 M guanidine-HCl. However, they are not soluble in non-ionic detergents (Table 1 ).
By comparing the electrophoretic motility of these forms with in vitro synthesized KAL, in the presence of 35S methionine (approximately 75 kDa; Fig. 2B), we predicted that they might be due to post-translational modifications. As six potential N-glycosylation sites are conserved between KAL and KALc proteins, we attempted to purify glycoproteins from COS 7 cell extracts by affinity binding to concanavalin A-Sepharose. In this way, the 100 kDa component of recombinant KAL and KALc proteins in the cell extracts was enriched, indicating that this form is due to glycosylation (Fig. 2C). The 85 kDa component may result from a different post-translational modification.
To answer the question whether or not KAL protein can diffuse in the extracellular matrix, we analyzed glycoproteins secreted in the conditioned medium of transfected cells by western blot. Both the ERB antisera and the 9E10 Mab detected a glycoprotein with an apparent molecular weight of approximately 45 kDa, in the conditioned medium of cells transfected with either the human or chick KAL cDNA (Fig. 2 D). This glycoprotein is consistently absent in the conditioned medium of mock-transfected cells (Fig. 2 D). Electrophoretic mobility of the 45 kDa component was the same under non-reducing conditions (data not shown). It is interesting to note that by using the KL antisera, we were not able to detect either this secreted 45 kDa glycoprotein or any other secreted protein in the conditioned medium of transfected cells. In addition, this low molecular weight diffusible component of KAL was never observed in cell extracts, suggesting that protein processing takes place on the cell membrane. Although we repeatedly observed this 45 kDa band in the conditioned medium of transfected COS cells, we cannot rule out the possibility that this is a proteolytic degradation occurring in cell culture conditions only.
To explore the cellular localization of KAL and KALc proteins, we performed immunofluorescence analysis on transiently transfected COS 7 cells. Using this system, when cell membranes were permeabilized with Triton X-100, both the anti-c-myc MAb 9E10 and the ERB antisera detected transfected cells because of their prominent endoplasmic reticulum and Golgi labeling (Fig. 3 A). This pattern is characteristic of a secretory protein. In addition, recombinant KAL was found to be secreted by transfected cells and to bind to the surfaces of adjacent untransfected cells (indicated by arrows in Fig. 3 A). Triton X-100 treatment does not extract the KAL protein from cell membranes, as supported by the conditions used for solubilization of KAL in western blot experiments (Table 1 ). Cell-surface localization was also confirmed on living transfected COS 7 cells using the ERB antisera (Fig. 3 B). No labeling was observed in mock-transfected cells (data not shown).
Gene transfection approaches proved useful for unravelling biosynthetic pathways of several cell adhesion molecules (34 ,35 ). By overexpressing the KAL and KALc proteins in COS 7 cells and using several antisera directed against the KAL protein, we have now identified KAL in western blot as a doublet of 100 and 85 kDa. The electrophoretic motility of these components is higher than that predicted by both the primary amino acid sequence and in vitro translation experiments. The 100 kDa component was shown to be the result of post-translational N-glycosylation. This finding is in agreement with the presence of six highly conserved N-glycosylation sites in the human and chick proteins. High salt, low pH, or chaotropic agents were necessary to extract these two components from cell membranes, whereas non-ionic detergents were ineffective, strongly suggesting that KAL might be a peripheral membrane protein. This is consistent with the absence of a membrane-spanning region in the protein. Several molecules with a role in neuronal migration and axonal targeting display linkage to the membrane through glycosyl-phosphatidylinositol (GPI) structures (28 ,36 ,37 ). However, KAL lacks a GPI-anchor sequence (38 ). KAL might bind to the cell membrane through electrostatic interactions or extracellular matrix components (39 ). Cell surface association was confirmed by immunofluorescence analysis on transfected cells.
In addition to being localized on the cell surface, KAL appears to be a secreted and diffusible molecule. KAL was detected in the conditioned medium of transfected COS cells and on cell surfaces of untransfected cells, indicating that it diffuses and attaches to adjacent cell membranes. Similar evidence was previously reported for F-spondin, a molecule secreted in the floor plate that promotes neural cell adhesion and neurite extension (40 ). Interestingly, we previously observed that in the human olfactory bulb, KAL mRNA is expressed in granule cells, while expression in chick appears confined to mitral cells (11 -14 ). The function of the diffusible form of KAL protein might, therefore, correlate with the incorporation into the extracellular matrix of the olfactory bulb in both species, regardless of the different cellular localization of the transcript.
KAL undergoes proteolytic cleavage to yield a diffusible component. Although we have observed this processing only in cell culture conditions, it is closely reminiscent of that previously demonstrated in cell adhesion molecules with FNIII repeats. The chick Bravo/NrCAM (41 ), neurofascin (42 ) and Ng-CAM (43 ) molecules and the mouse L1 (44 ) protein have been shown to be heterodimers composed of [alpha]- and [beta]-chains. These chains are derived in vivo from an intact precursor polypeptide by proteolytic cleavage. Cleavage in Nr-CAM, Ng-CAM and L1 occurs at comparable sites in the middle of the third FNIII repeat, whereas the neurofascin molecule is cleaved between the immunoglobulin domains and the FNIII repeats. In each case, proteolytic cleavage occurs within paired dibasic sequence motifs. Such motifs have been shown to be endoprotease recognition/cleavage sites for a large number of preproteins (45 ,46 ). By using ERB antisera, we were able to distinguish a 45 kDa glycoprotein in the conditioned medium of transfected cells.
A strong site of recognition for a trypsin-like protease is the sequence KKKRRK at positions 345-350, within the second FNIII repeat of the human KAL protein. This sequence is perfectly conserved in the chick KALc protein. Sequence alignment of the second FNIII repeat of KAL and KALc with the third FNIII repeats of Bravo/Nr-CAM, Ng-CAM and L1 shows that the KKKRRK sequence may correspond to the paired dibasic amino acid stretch in which cleavage occurs (Fig. 4 ). Cleavage within a KKKKK sequence has also been proven for LAR, a receptor-linked protein tyrosine phosphatase with FNIII repeats (46 ). Cleavage within the KKKRRK sequence would explain why we could not detect either the secreted 45 kDa KAL protein or any other diffusible form using the KL antisera. The KKKRRK sequence is in fact situated within the synthetic peptide used to raise the KL polyclonal antibodies. Furthermore, cleavage within the KKKRRK sequence would yield a C-terminal fragment whose size is compatible with the 45 kDa fragment, taking into account the presence of three N-glycosylation sites.
The region of KALc from nucleotides 1464 to 2117 (corresponding to amino acids 456-672) was amplified using specific primers and cloned in frame with the carboxy-terminus of the glutathione S-transferase (GST) from Schistosoma japonicum in the pGEX-3X plasmid vector (Pharmacia). DH5[alpha]F' competent cells were transformed with the construct and several colonies were grown, induced with IPTG and tested for overproduction of the GST-KALc fusion protein on a 10% SDS-PAGE gel. Isolation of inclusion bodies was performed by centrifugation, coupled with washing procedures (47 ). Subsequently, the GST-KALc fusion protein was electroeluted from a 10% SDS-PAGE gel using a Biorad model 442 electroeluter. Removal of SDS from the buffer was achieved by several steps of dilution in PBS and concentration on a Centricon 30 column (Amicon).
A 15 amino acid synthetic peptide corresponding to positions 340-354 of human KAL protein and conjugated to the MAP resin, was obtained through the Protein Chemistry Core Facility of Baylor College of Medicine, Houston, TX. Rabbit polyclonal antibodies were generated against both the GST-KALc fusion protein (ERB) and the synthetic peptide (KL) by PEL-FREEZR, Rogers AR. Total IgG were purified using HiTrap Protein A columns (Pharmacia), according to the manufacturer's instructions.
PCR with specific oligonucleotides was used to introduce the coding region of the KAL and KALc cDNAs into a modified pMT21 vector containing the c-myc epitope (a kind gift from Marc Tessier-Lavigne), to yield expression of a c-myc-tagged KAL and KALc proteins. Transient transfections of pMT21-KALmyc and pMT21-KALcmyc into COS-7 cells were performed using lipofectAMINE (GIBCO, BRL), as directed.
To prepare cell extracts, cell monolayers were incubated, after removal of media, with 1 M NaCl, 50 mM Tris-HCl pH 8, 1* protease inhibitors and incubated at room temperature for 5 min. Extracts were then incubated on ice for 30 min and centrifuged for 30 min at 14 000 r.p.m. in a microfuge. The same amount of acetone-precipitated proteins were subjected to western blot analysis.
In order to isolate glycoproteins, cell extracts were diluted 1:10 in concanavalin A buffer (0.1 M acetate buffer pH 6, 1 M NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2) and incubated with concanavalin A-Sepharose 4B (Sigma) overnight at 4oC. After five washes with concanavalin A buffer, concanavalin A-Sepharose pellet was resuspended in SDS-PAGE loading buffer. Glycoproteins were released by boiling for 10 min and subject to western blot analysis. Cell culture media were concentrated into Centriprep 30 (Amicon) and diluted 1:10 with concanavalin A buffer. Isolation of glycoproteins was then carried out as described.
Primary antibodies were used at 1 [mu]g/ml (ERB) to 2 [mu]g/ml (KL) of total IgG, whereas Mab 9E10 (32 ) supernatant was diluted 1:50. Visualization of antibody binding was carried out with Enhanced ChemiLuminescence (Amersham Corp.), according to the manufacturer's instructions.
KALc tagged with the c-myc epitope was detected with both the Mab 9E10 (neat tissue culture supernatant) (32 ) and the ERB antisera (10 [mu]g/ml of total IgG). Fluoresceinated isotype-specific secondary antibodies (DAKO; rabbit antimouse and porcine antirabbit IgG) were used at a dilution 1:100.
For immunofluorescence membrane labeling, cultures were washed once at 22oC with DMEM medium without serum and then incubated with primary antibody for 30 min at 22oC in DMEM/10% porcine serum. Cultures were then washed twice with DMEM and incubated with secondary FITC-conjugated isotype-specific antibody diluted in DMEM/10% porcine serum for 30 min at 22oC. Cultures were washed twice and fixed in 4% paraformaldehyde/PBS for 20 min, rinsed in PBS and coverslipped in Vectashield (DBA).
For intracytoplasmatic antigen detection, cultures were washed once at 22oC with DMEM without serum and fixed in 4% paraformaldehyde/PBS for 20 min. Cultures were then rinsed with PBS and incubated in 0.2% Triton X-100/PBS for 30 min at 22oC, in order to permeabilize cell membranes. Cultures were washed twice with PBS and incubated with 10% porcine serum/PBS for 1 h. After two washes with PBS, incubation with primary antibody diluted in 0.1% Triton X-100/10% porcine serum/PBS was performed for 4 h at 22oC in a humid chamber. Cultures were then washed twice with PBS and incubated with secondary FITC conjugated isotype-specific antibody diluted in 0.1% Triton X-100/10% porcine serum/PBS for 30 min at 22oC. Finally, cultures were washed twice with PBS and coverslipped in Vectashield (DBA). Cultures were viewed on a Zeiss Axioplan microscope under epifluorescence optics.
In vitro synthesized KAL protein was generated in the presence of 35S methionine by means of the Rabbit Reticulocyte Lysate System (Amersham), using in vitro-transcribed RNA as a template, obtained with the RNA Transcription kit (Stratagene). The product was analyzed by SDS-PAGE and visualized by autoradiography.
We thank Drs Alessandro Guffanti, Germana Meroni, Ruggero Pardi and Vittoria Schiaffino for helpful discussion and Ms Melissa Smith for help in preparation of the manuscript. This work was supported by grants from the March of Dimes Birth Defects Foundation and the Italian Telethon Foundation.
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