Rapid Communication: Characterization of ?Amyloid Peptide from Human Cerebrospinal Fluid

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Abstract: β-Amyloid peptide (Aβ) is one of the main components of senile plaques in the brain tissue of Alzheimer's disease (AD) patients. Aβ is proteolytically cleaved from the amyloid precursor protein (APP), an integral membrane protein
  Journal zyxwvusrqpo   zyxwvusrqponm eurochemisfry zyxwvusrqpon aven Press, Ltd., New zyxwvutsrqpo ork zyxwvutsrq   993 International Society for Neurochemistry Rapid Communication Characterization of P-Amyloid Peptide from Human Cerebrospinal Fluid Carmen Vigo-Pelfrey, Doris Lee, Pam Keim, Ivan Lieberburg, and Dale B. Schenk Athena Neurosciences, Inc., South San Francisco, California, U.S.A. zyx Abstract: zyxwvutsrqpon -Amyloid peptide (AD) is one of the main components of senile plaques in the brain tissue of Alzheimer s disease (AD) patients. APis proteolytically cleaved from the amyloid precursor protein (APP), an integral membrane protein possessing a large extracellular N-terminal domain followed by a single membrane- spanning region and a short cytoplasmic C-terminal tail. AP has been isolated rom senile plaques and cerebral vascular tissue of AD brain and characterized as a heterogeneous peptide contain- ing 28-43 amino acids whose sequence begins in the extracellu- lar domain of APP and extends into the putative transmembrane sequence. It has long been speculated hat AP may also be pres- ent in body fluids, such as CSF, that contact neuritic plaques. Recently using a specific enzyme-linked immunosorbent assay we were able to quantify one form of A6 in CSF. In this report, using one of these antibodies covalently bound as an affinity ma- trix, multiple complex forms of AD have been isolated and charac- terized from CSF derived from patients with either meningitis or other neurological disorders. Amino acid sequencing reveals AP species with N-termini of Asp , Glu3 His6, Glu , and Val , al- though on a molar basis, Asp represents he predominant amino- terminus. Laser desorption mass spectrometry confirmed the presence in CSF of A0 species containing 27, 28, 30, zyxwvut 4 5, 40 42 nd 43 amino acids, all beginning at Asp ; two stable trimers, (A~p -Met~~), nd (Hi~ -Ala~ )~; nd one stable dimer containing (A~p -Val~~),. ome of these fragments have yet to be identified in brain either because they are generated solely in the CSF used in this study or because current procedures used to isolate brain amyloid result in their loss. Key Words: 0-Amyloid peptide-Alz- heimer s disease-Human GSF. zyxwvutsrq   Neurochern. 61,1965-1968 (1993). P-Amyloid peptide (AD) has been isolated from brain and the cerebral blood vessels of patients with Alzheimer's dis- ease (AD) (Glenner and Wong, 1984a,b; Masters et al., 1985; Gorevic et al., 1986; Roher et al., 1986; Selkoe et al., 1986; Joachim et al., 1988; Prelli et al., 1988a,b; Miller et al., 1990), trisomy 21 (Down's syndrome) (Glenner and Wong, 1984a,b), and hereditary cerebral hemorrhage with amyloidosis-Dutch type (van Duinen et al., 1987; Prelli et al., 1988a,b) and from undiseased aged brain (Coria et al., 1987). The basics of purification have always relied on the extreme insolubility of Ap-a point that will be readdressed later. Over the past 8 years several groups have attempted to measure A@ roduction and release into body fluids in vivo and in vitro (Wong et al., 1985; Selkoe, 1986, 1989; Par- dridge et al., 1987a,b; Joachim et al., 1989; Mehta et al., 1991). A complicating factor in this regard is that the se- creted form of amyloid precursor protein (APP) contains the first 15 amino acids of the AD peptide at its carboxy-ter- minus, leading potentially to immunologic cross-reacting species. Direct attempts to demonstrate the presence of Ap in human CSF have been unsuccessful until recently (Seu- bert et al., 1992; Shoji et al., 1992). We have undertaken the present work to show unequivocally that AD is present in CSF and to more fully characterize it. MATERIALS AND METHODS CSF source Three liters of CSF obtained from >3,000 individuals with various medical conditions was obtained from Univer- sal Reagents. Seventy percent of the sample consisted of suspected meningitis (of which 20 was juvenile and 80% adult), and the remaining 30% was derived from various neurological disorders (including multiple sclerosis, cerebro- vascular accident, demyelinating disease, hypertension, de- mentia, and migraine headache). Half of the CSF individual samples were centrifuged by the laboratory to separate po- tential peripheral blood elements, and all of the material was filtered (pore size, 0.45 pm). Absorbance measurements of the CSF pool gave a reading of ~0.02 nits at 405 nm, indicating the hemoglobin content to be 55 pg/ml (Crosby et al., 1954), suggesting the absence of detectable hemolysis or cellular contamination in the sample tested. The individ- ual samples were pooled and kept at -70°C until use. Affi- Gel Hz Hydrazide resin was obtained from Bio-Rad. Monoclonal antibodies The monoclonal antibody 266 was generated against the synthetic peptide HHQKLVFFAEDVGSNKGGC (ADl3& conjugated to a-CD3 (Boehringer Mannheim) via m-malei- midobenzoyl-N-hydroxysuccinimide ster (Pierce) before injection into A/J mice as previously described (Seubert et al., 1992). Resubmitted manuscript received July 19, 1993; accepted July 23, 1993. Address correspondence and reprint requests to Dr C. Vigo-Pel- frey at Athena Neurosciences, Inc., 800F Gateway Boulevard, South San Francisco, CA 94080, U.S.A. Abbreviations used: AD P-amyloid peptide; AD, Alzheimer's dis- ease; APP, amyloid precursor protein. 1965  C. VIGO PELFREY ET AL. 966 zyxwvusr 160 zyxwvutsrqponmlkjih 14 zyxwvutsrqponmlkjihgfedc 120 zyxwvutsrqponmlkjihgfed oo - 2 00 E - zyxwvutsrqponmlk 0 40 20 30 35 40 45 zyxwvutsrqponml 0 55 60 65 70 Fraction Number FIG 1 HPLC chromatogram of the CSF material bound by the AP-affinity column eluted by 0.2 glycine, pH 2. zyxwvuts Preparation of affinity column The monoclonal antibody 266 was coupled to Affi-Gel Hz Hydrazide resin according to the manufacturer's in- structions. Isolation of A@ rom CSF Three liters of CSF was filtered through a 450-nm-dia- meter sterile filter and affinity-purified using the monoclo- nal antibody 266 covalently bound to Affi-Gel Hz resin. Filtered CSF was passed through the resin at -3 mlfmin at 4°C. All detectable immunoreactivity present in CSF (2.5 ng/ml) was completely depleted by passage through the af- finity column. The resin was then washed with 200 ml of phosphate-buffered saline, and the specifically bound mate- rial was eluted in 20 I-ml fractions with 0.2 Mglycine, pH 2.0. The fractions were then assayed for A@ mmunoreactiv- ity, and six positive fractions were pooled and further puri- fied by HPLC. The pooled material was subjected to re- verse-phase HPLC using a Vydac C4 0.2- X 15-cm) column as previously described (Seubert et al., 1992). The A/3 recov- ery of the various A@ peptides used as internal standards ranged from 30 to 40%. The fractions positive for A0 in the ELISA were subjected to microsequencing and to laser de- sorption mass spectrometry. A microsequence The peptides eluting in the various fractions collected from HPLC were then microsequenced on an Applied Bio- system model 477 protein sequencer using a microscale reac- tion cartridge and Applied Biosystem's MICFST program cycles. Determination of the molecular masses of A@- positive fractions The molecular masses ofthe peptides eluting in the peaks shown in Fig. 1 were determined by matrix-assisted, laser desorption time-of-flight analysis using a-cyanohydroxy- cinnamic acid as a matrix in a Finnigan Lasermat mass spectrometer. (Mass analyses were performed by the Bio- technology Instrumentation Facility, University of Califor- nia, Riverside.) RESULTS Purification of A@ rom CSF Three liters of CSF was totally depleted of A@ mmunore- activity after passage through the affinity column, as deter- mined by the A@ ELISA. The eluted material was further purified by HPLC (Fig. 1 . Peptides eluting in peak fractions of the chromatogram shown in Fig. 1 were then analyzed for TABLE I. N-Terminal sequence data for all fructions showing sign$cant AP immunoreactivity in the ELISA AP Fraction Initial ELISA no. N-Terminal sequence yield reactivity' 32.33 35, 36 41 46 52 55 58 61 62 64,65 XXEFR HDS(G)Y XXXXY DAEFR HDSGY XXEFX XXEFX DAEFR HDSGY XXSXY XXEFR HDSGX DAEFR HDSGY Sequence undetermined XQEFP QD(S,Q) DAEFR HDSGY XXSXY KL(V)FF KLXFF XLX(F)F KLVFF KLXFF KLVFF X XLX (E)X KLVFF KLXFF AE(D)X (A)EDX (A)(E)(D)XG AEDVG AEX AXDXG AEDVG AEDX(G) 5.3 SNX 3.9 16 <I <I SXXGX 150 20 sx 49 4.5 10.7 1.8 SNX 102 WNW 4.6 zy   + All fractions showing significant A0 immunoreactivity in the ELISA were then microsequenced. All of them, with the exception of fractions 61 and 62, also yielded N-terminal sequence data corresponding to A& X, residue not identified. Parentheses indicate uncertainty of amino acid assigned. Data are from a single analysis of each HPLC fraction. As determined by protein sequencing, the total of AD species detected in the HPLC fractions was -370 pmol (the sum of values here). In a separate experiment, when 1 fig of synthetic A0 1-40 was applied to the C, column, -30 was recovered. Assuming 30% as the average recovery of all A@ pecies, 50% as the initial sequencing yield, and 4 kDa as the average molecular mass, then the total mass of AP-related peptides from 3 L of CSF is - 0 zyxwvutsr g J. Neurochem. Vol. 61 No 5 1993  CHARACTERIZATION OF 0-AMYLOID PEPTIDE FROM HUMAN CSF 1967 TABLE 2. Further characterizalion offractions showing zyxwvut P immiinoreaclivily using laser alomic desorption mass spectrometry zyxwvuts ~~~~ zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA HPLC Sequence Mass Calculated fraction no. observed observed Fragment mass 32,33 35,36 41 46 52 55 58 61 62 64,65 Asp' His6 Asp' Asp' Asp' Asp' His6 Glu Asp' Asp' zyxwvutsr   ? Asp' 3,389 3,262 2,516 3,134 3,443 3,298 3,603 3,791 3,173 2.634 3,931 3,315 4,138 8,640 3,800 I 1,642 5,8 1 gd 11,738 S,88Se 4,337 1-30 1-28 6-27 1-27 11-43 6-3.5 3-34 1-34 6-34 1 1-34 1-35 12-43 1-38 1-40 (d)b 1-34 6-42 (t)'. 1-35 (t)b.' 1-40 3,394 3,266 2,521 3,138 3,440 3,303 3,604 3,790 3, I72 2,613 3,921 3,311 4,134 8,662 3,790 1 1.694 11,763 4.332 The molecular masses of all the A6 positive peaks shown in Fig. 1 ata are from a single mass analysis of each HPLC fraction. are summarized. d, dimer; t, trimer. These assignments are made strictly on the basis of best mass Probably the doubly charged ion of mass 11,642. Probably the doubly charged ion of mass I 1,738. fit. Other interpretations are possible. AP immunoreactivity. All the peaks showing AP immunore- activity were then microsequenced. AD microsequence from CSF All fractions (with the exception of fractions 6 1 and 62) showing significant reactivity in the ELISA also yielded N- terminal sequence data corresponding to AP (Table 1). In fraction 52 a previously described AP fragment starting with Glu (Seubert et al., 1992) and a novel A@ ragment starting at His6 were also found in peaks 64 and 32. Fractions 6 1 and 62 did not yield any significant AP sequence despite possess- ing high AD immunoreactivity. It is possible that these frac- tions contained a blocked amino-terminal form of AD simi- lar to those found in AD neuritic plaques (Mori et al., 1992). Determination of molecular mass of A@ rom CSF Using laser atomic desorption mass spectrometry, we fur- ther characterized and estimated the molecular mass and length of all the various immunoreactive peaks separated by HPLC. Table 2 summarizes the molecular masses of all the different A@ species found in CSF. DISCUSSION The present results unequivocally demonstrate the pres- ence of AP in CSF obtained from suspected meningitis and various neurological disorders. Furthermore, AP was found to be in multiple forms, comprising AP peptides of various lengths with -70% of the material composed of and AP,-,,,. These data suggest that this peptide undergoes lim- ited proteolysis or, alternatively, that these fragments could be derived from differential secretory cleavage of APP. Se- creted A@ was detected in CSF, but no full length-APP in CSF was detected before or after affinity purification. Similar but distinct AP heterogeneity has been also found in neuritic plaques of the Alzheimer's type. Characteriza- tion of cerebrovasculature amyloid has consistently yielded AP fragments of amino acids 1-39, 1-40, 1-42, and 1-43, but amyloid isolated from neuritic plaques has given mixed data (Bouman, 1934; Glenner and Wong, 1984a,h; Masters et al., 1985; Mortimer et al., 1985; Kang et al., 1987; Joa- chim et al., 1988; Prelli et al., 1988a,b). Plaque-derived amyloid has also exhibited both N- and C-terminal heteroge- neity. Two reports have suggested that the predominant C- terminus is at position 42 (Roher et al., 1993), whereas an- other report identified position 40 (Mori et al., 1992); a third one showed that peptide 1-43 was the predominant form in cortical microvessels (Pardridge et al., 1987b). The results published so far refer to the AP composition in the plaques of AD patients, but our results reflect the AP com- position in non-AD CSF derived from various neurological states. However, inherent in all of these previous studies are difficulties n interpreting yields of peptides because the iso- lation procedures used all require homogenization, deter- gent treatment, and centrifugation and therefore might re- sult in selective enrichment of only a subset of the most resistant amyloid fibrils (Frucht et al., 1992). In this light, it will be informative to reexamine the nature of brain amy- loid by isolating it using immunoaffinity techniques to im- prove potentially the yield of AP fragments. In addition, at this point it is not clear whether there is any selectivity of AP forms deposited in the plaque and CSF, or whether this process is indiscriminate. It is possible that there is a continuous exchange and par- titioning of AP between plaques and CSF. Recent data in support of this hypothesis were obtained by Maggio et al. (1 992), who showed that radiolabeled AP binds to plaques in the nanomolar range in AD brain tissue. From the pres- ent data it is unclear why multiple forms of AP are found in CSF, or whether this process is time dependent. Further- more, the antibodies used do not recognize small AP frag- ments, that would not have been retained by the affinity column and consequently not sequenced. The existence of these fragments therefore cannot be excluded, and the heter- ogeneity reported in our studies may be even greater. Such heterogeneity might also suggest the action of soluble pro- teases, resulting in the ragged N- and C-termini species found in CSF. However, if such a proteolytic process exists, it is clearly not limited to CSF, because in plaques of AD and Down's syndrome patients, AP species starting with Glu3, Phe4, Ser*, Leu , and Gly29 have also been identified (Masters et al., 1985; Mori et al., 1992). Nevertheless, we cannot exclude the possibility that the AP fragments that we have characterized in this study are different from those present in healthy control or AD CSF. Relevant to this, AP fragments have recently been fully characterized from con- ditioned media of APP-transfected 293 cells with findings very similar to those reported here that AP,-,, and AP,-40 re the two most common fragments (Dovey et al., 1993). TO address these issues fully, AP will ultimately need to be iso- lated from AD and control CSF to compare differences care- Our results unequivocally demonstrate that soluble AP peptides are present in CSF at low concentrations. It is possi- ble that the process of plaque formation and AP deposition fully. zyxw f Neurorhem., Vol. 61. Nu. zy   1993  I968 C. VIGO-PELFREY ET AL. that occurs during aging and zyxwvutsrq D results in quantitative changes of this peptide in CSF. The loss of neurons that occurs in AD brain might also be a result ofthis process. It is therefore very important to analyze A0 in CSF, to under- stand both its normal physiology and its pathophysiology as seen in AD and other neurological disorders. It is hoped that this effort will help to clarify the mechanisms responsible for zyxwvutsrq D deposition and provide diagnostic and prognostic utility. Acknowledgment: We are grateful to K. Springer from Universal Reagents for providing us with the CSF pool and important information regarding the nature and handling of CSF: J. Whaley, C. Swindlehurst, and B. Wolfert from Hybritech for providing us with the 266 junction antibod- ies: to the University of California, Riverside, Biotechnol- ogy Instrumentation Facility for mass spectrometry analy- sis; P. Seubert for preparing the affinity resin; and T. David- son for typing this manuscript. REFERENCES Bouman L. zyxwvutsrqpon   I9 34) Senile plaques. Bruin 57, 128- 142. Coria F.. Castano E. M., and Frangione B. (1981) Brain amyloid in normal aging and cerebral amyloid angiopathy is antigenically related to Alzheimer’s disease beta-protein. zyxwvutsrq m. J. Puthol. 129, 422-428. Crosby W. M.. Munn J. J., and Furth F. W. 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