Proteomic analysis shows that the main constituent of subepidermal localized cutaneous amyloidosis is not galectin-7.

Abstract

Lichen or macular localized cutaneous amyloidoses have long been described as keratinic amyloidoses and believed to be due to the deposition of cytokeratin peptides originating from epidermis in the dermal papillae. However, recently it was suggested that galectin-7 is the causative protein for this type of amyloidosis. This was based on the detection of galectin-7 in a biopsy from a patient diagnosed with Bowen’s disease and localized cutaneous amyloidosis. In this study we report mass spectrometry-based proteomic analysis of the protein composition of localized cutaneous amyloid deposits from seven patients using laser microdissection and show that basal keratins are the main constituents of the amyloid deposits. Galectin-7 was not present in the dermal amyloid deposits and was only present in the overlying Congo red negative epidermis.

Introduction

Cutaneous amyloidosis can be localized or systemic with secondary cutaneous involvement [1,2]. In both cases insoluble amyloid fibrils composed of amyloidogenic protein are deposited in the extracellular regions of the skin. The most common systemic amyloidoses associated with secondary cutaneous lesions are AL amyloidosis and AA amyloidosis [3]. Systemic amyloidosis deposits are typically located in blood vessel walls in the dermis [4].

Localized cutaneous amyloidosis includes so-called lichen or macular and primary nodular forms. Localized nodular cutaneous amyloidosis is predominantly AL amyloidosis caused by deposition of immunoglobulin light chains produced by a local B-cell or plasma cell disorder and present as yellowish waxy nodules [5].

Lichen or macular amyloidosis are terms that refer to the clinical appearances of the diseases, but deposits are histopathologically identical. Deposits are predominately found in the papillary dermis directly below the basal epidermal layer without blood vessel involvement [6]. Localized cutaneous amyloidosis usually presents with itching and visible changes in skin pigmentation and thickening.

Lichen or macular localized cutaneous amyloidoses have long been described as keratinic amyloidoses and believed to be due to the deposition of cytokeratin peptides from degenerating epidermal cells [7]. The keratin gene family consists of 54 distinct functional genes, but keratin expression is dependent upon the cell type, differentiation state of the cells, and can be modulated upon damage or infection [8]. Cytokeratins are expressed in the cytoplasm of epithelial cells. Proliferative basal keratinocytes are mitotically active and express keratin 5, 14, and small amounts of 15. Upon differentiation these cells stop dividing and start expressing keratin 1 and 10 as they migrate into the suprabasal layers. Immunohistochemistry (IHC) and immunofluorescence studies have shown that keratin 5 is the predominant cytokeratin protein in amyloid deposits and keratin 14 is less commonly localized to amyloid deposits [9,10].

Extensive studies over many years supported that the formation of localized cutaneous amyloid deposits started with the damage of keratinocytes that leads to degeneration of the cells and formation of filamentous masses composed of cytokeratins that drop into the dermis [7,11]. However, in 2013 Miura and colleagues proposed that galectin-7 was the protein responsible for lichen or macular localized cutaneous amyloidosis [12]. Galectin-7 is a member of the galectin protein family of lectins, which contribute to the differentiation and development of stratified epithelia [13]. It is also involved in the epithelialization of epidermal wounds.

This conclusion was based on the biochemical analysis of a biopsy from a patient with Bowen’s disease associated cutaneous amyloidosis including Western blots and MALDI mass spectrometric. Serum amyloid P, apolipoprotein E, cytokeratins, actin and galectin-7 were identified. The localization of galectin-7 to the amyloid deposit was determined by comparing the Congo red stain and IHC with anti-galectin-7 antibodies. In 2012 AGal7 was preliminarily added to the amyloid fibril proteins and amyloidosis list by the Nomenclature Committee of the International Society of Amyloidosis (ISA) and was kept on the official list during more recent committee reviews [14–16].

In this study we performed high resolution analysis of localized cutaneous amyloidosis using laser microdissection (LMD) and liquid chromatography-mass spectrometry (LCMS) to determine the protein composition of localized cutaneous amyloid deposits as compared to the surrounding tissue [17]. Laser microdissection allows for precise collection of the amyloid deposits with minimal contamination from the surrounding tissue as has been widely shown through its application to clinical diagnosis [18,19]. When paired with the sensitivity of mass spectrometry we can confidently determine the protein composition of the cutaneous amyloid deposits as compared to the surrounding dermis and overlying epidermis. Here, we report the results from the analysis of specimen from five patients diagnosed with localized cutaneous (lichen or macular) amyloidosis and two patients diagnosed with nodular cutaneous amyloidosis.

Material and methods

Formalin-fixed paraffin-embedded (FFPE) skin biopsy samples from seven patients diagnosed with localized cutaneous amyloidosis were used for this study (Table 1). Patients ranged from 36 – 71 years of age and were predominately male. Five of the patients (patients 1 – 3 and 6 – 7) had a diagnosis of localized cutaneous macular or lichen amyloidosis and two patients with localized nodular cutaneous amyloidosis were included as controls (patients 4 – 5).

Table 1.

Localized cutaneous amyloidosis patient demographics.

Patient	Gender	Age	Tissue	Diagnosis
1	M	50	Skin, Forehead	Amyloid with atypical perivascular lymphoid infiltrate
2	M	64	Skin, Shin	Seborrheic keratosis with papillary dermal amyloid deposition
3	M	67	Skin, Leg	Lichen amyloidosis
4	F	36	Skin, Back	Nodular amyloid deposition
5	F	57	Skin, Back	Interstitial and nodular amyloid deposition associated with plasma cell infiltrate
6	M	51	Skin, Back	Primary cutaneous amyloidosis
7	M	71	Skin, Pubic	Papillary dermal amyloidosis

For each FFPE biopsy, sections 10 μm thick were placed onto DIRECTOR™ slides (Expression Pathology) and stained with Congo red. Congo red positive tissue areas were identified by bright-field and fluorescence microscopy (Figure 1). A total of 50,000 μm² of Congo red positive tissue was microdissected into 0.5 ml microcentrifuge tube caps containing 10 mM Tris/1 mM EDTA/0.002% Zwittergent 3–16 (Calbiochem) using a Leica LMD 6500 Microdissection System (Leica Microsystems) [17]. Overlying intact epidermis (Congo red negative) areas totalling 50,000 μm² were laser microdissected from each section as above (Figure 1). Tissue samples were heated at 95°C for 90 minutes, vortexed once after 45 minutes, followed by 60 minutes of sonication in a waterbath. Samples were digested overnight at 37°C with 1 μg of trypsin (Promega) and then desalted using C18 spin columns (Harvard Apparatus).

Figure 1.

Images of the laser microdissected regions of tissue visualized in brightfield and fluorescence microscopy. Examples of the regions microdissected are shown for patient 3 (A – D) and patient 1 (E – H). All specimen were viewed by brightfield (A, C, E, G) and fluorescence (B, D, F, H) microscopy before microdissection. A total of 50,000 μm2 of either Congo red positive or neighbouring negative tissue was microdissected. All images show the red or green outlined regions that were microdissected.

Liquid chromatography-mass spectrometric analysis of the digested tissue samples was performed on an UltiMate™ 3000 RSLCnano and Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) as previously described [19–21]. Data was searched using Byonic within Proteome Discoverer (Thermo Fisher Scientific) against the UniProt human reference database with the addition of immunoglobulin variant domains and common contaminant protein sequences. Only high confidence peptide spectral matches (PSMs) with FDR of 1% or less and a Byonic score of 300 or higher are reported. Proteins with at least one unique peptide were considered for interpretation.

IHC was performed on formalin fixed paraffin embedded tissue using monoclonal mouse antibody 34ßE12 (Roche Diagnostics) for keratins 1, 5, 10, and 14 on a Roche Benchmark Ultra (Roche Diagnostics). Antigen retrieval was performed with Cell Conditioning Solution 2 (Roche Diagnostics) for 24 minutes, antibody was used as received and incubated for 28 minutes at room temperature. The Optiview detection kit (Roche Diagnostics) was used for detection. IHC for galectin-7 utilized the monoclonal rabbit antibody EPR4287 (Abcam) on a Leica Bond III (Leica Biosystems). Epitope retrieval was performed with Leica Epitope Retrieval Solution 2 for 30 minutes. The antibody was diluted 1:4K with Lab Vision Antibody Diluent OP Quanto (Thermo Fisher Scientific), incubation was performed at 25° C for 30 minutes, and the Polymer Refine Detection kit DS9800 was used (Leica Biosystems).

Results

The peptide profile identified by mass spectrometric analysis of the microdissected dermal amyloid deposits from the five patients with localized cutaneous lichen or macular amyloidosis were consistent with keratinic amyloidosis [9,22]. Specifically, keratin 5 was abundant and keratin 14 was also present in the microdissected amyloid deposits. Additionally, the common amyloidosis protein markers identified in all subtypes, apolipoprotein E and serum amyloid P, were detected in the amyloid deposits. Galectin-7 was not detected in any of the Congo red positive dermal regions. Galectin-7 was only detected in the overlying Congo red negative epidermis microdissected from the same biopsies (Table 2). Serum amyloid P and apolipoprotein E were not seen in the Congo red negative epidermis. Actin was present in both the amyloid deposits and the overlying unaffected epidermis at comparable levels.

Table 2.

Results of the mass spectrometric analysis of tissue microdissected from localized cutaneous amyloidosis patient biopsies. The number of peptide spectral matches per protein for each patient’s Congo red positive (amyloid deposits) and negative (overlying epidermis) samples are shown. Apolipoprotein E and serum amyloid P are general marker proteins detected in amyloid deposits and are displayed in the upper part of the table. The center segment contains results for causative (theoretical or confirmed) amyloid proteins and the bottom contains two common contaminant keratin proteins and actin. Peptide spectral matches (PSMs) can be used to determine the relative abundance of a protein. Amyloid protein markers serum amyloid P and apolipoprotein E are detected in all Congo red positive amyloid deposit samples and in none of the Congo red negative samples. Galectin-7 is only detected in the Congo red negative overlying epidermis samples. (LCA = primary localized cutaneous amyloidosis)

Protein Name	Gene Symbol	Protein Accession	# PSMs
			1		2		3		4		5		6		7
			LCA		LCA		LCA		AL Lambda		AL Lambda		LCA		LCA
			Amyloid Deposits	Overlying Epidermis	Amyloid Deposits	Overlying Epidermis	Amyloid Deposits	Overlying Epidermis	Amyloid Deposits	Overlying Epidermis	Amyloid Deposits	Overlying Epidermis	Amyloid Deposits	Overlying Epidermis	Amyloid Deposits	Overlying Epidermis
			Amyloid Assosicated Proteins
Apolipoprotein E	APOE	P02649	5	0	12	0	17	0	11	0	37	0	15	0	11	0
Serum Amyloid P	SAMP	P02743	9	0	19	0	22	0	10	0	12	0	17	0	9	0
			Amyloid Subtype Proteins
Keratin 14	KRT14	P02533	5	58	19	37	61	173	2	37	0	17	17	22	11	8
Keratin 5	KRT5	P13647	12	45	22	37	53	42	0	63	0	16	32	14	27	19
Ig lambda-2 chain C	IGLC2	P0CG05	0	0	0	0	0	0	12	0	36	0	0	0	0	0
Ig lambda chain V-III region LOI	IGLV3-21	P80748	0	0	0	0	0	0	0	0	6	0	0	0	0	0
Ig lambda chain V-III region SH	IGLV3-19	P01714	0	0	0	0	0	0	18	0	0	0	0	0	0	0
Ig gamma-1 chain C	IGHG1	P01857	0	0	0	0	4	0	2	0	28	0	4	0	2	0
Galectin 7	LGALS7	P47929	0	16	0	7	0	24	0	18	0	6	0	7	0	5
			Common Keratin Contaminants & Actin
Keratin 1	KRT1	P04264	11	93	7	84	30	76	25	69	8	19	15	28	19	24
Keratin 10	KRT10	P13645	15	111	8	103	5	58	36	62	5	7	31	7	13	21
Actin	ACTB	P60709	5	8	2	5	18	13	3	7	10	2	7	2	11	2

As expected, the peptide profile identified in the amyloid deposits microdissected from the localized nodular cutaneous amyloidosis were consistent with AL amyloidosis (Table 2) [17–19,23–25]. In addition to apolipoprotein E and serum amyloid P, lambda light chain constant region and variable region peptides were abundant in the amyloid deposits confirming immunoglobulin light chain as the causative protein (Table 2). The presence of a plasma cell or B-cell disorder was confirmed by histopathological analysis. Galectin-7 was not detected in the amyloid deposits of the nodular cutaneous patient biopsies. However, Galectin-7 was detected in the overlying Congo red negative epidermis. In addition, keratins 5 and 14 were not detected in the nodular cutaneous amyloid deposits of patient 4. Keratin 5 was not detected in patient 5 and keratin 14 was only detected at a very low level (fewer than 5 peptide spectral matches). Actin was present in both the amyloid deposits and the overlying unaffected epidermis microdissected from both nodular cutaneous amyloidosis patients at comparable levels.

Galectin-7 was detected with 45 – 62 % sequence coverage in all Congo red negative overlying tissue samples (Figure 2). When corrected to only consider peptides theoretically detectable by LCMS the sequence coverage of galectin-7 is as high as 80%. Additionally, all detected galectin-7 peptides are unique to this protein sequence [26]. The sequence coverage of keratin in the localized cutaneous lichen or macular amyloidosis varies between 11 – 49% and all peptides identified are unique to keratin 5 or keratin 14 (Table S1). Of note, the sequence coverage of keratins 5 and 14 is higher in the overlying dermis tissue than in the amyloid deposits. In supplemental figure 1, a comparison of the keratins 5 and 14 peptides identified in both tissue types are shown.

Figure 2.

Galectin-7 mass spectrometry sequence coverage was 45 % - 62 % in Congo red negative samples. Galectin-7 peptides detected by LCMS in the Congo red negative tissue microdissected from each specimen are indicated by the presence of a black line. Each sample is indicated by the number on the left end of each sequence line. Galectin-7 was not detected in any of the Congo red positive tissue samples.

Previous studies of other amyloidosis subtypes, primarily in adipose tissue, have shown that amyloid deposits commonly contain truncated or shortened forms of proteins [27–30]. The cytokeratins may be truncated or fragmented prior to or during deposition or fibril formation and trypsin digestion of fragmented protein would not result in the peptides predicted from a theoretical digest of the intact protein sequence. However, keratin 5 and 14 in the overlying dermis tissue is likely the intact form the protein and when digested would produce predictable tryptic peptides.

Sequence homology of cytokeratins is very high, but when the proteins are digested into peptides by trypsin they generate many unique peptides. In the amyloid deposits the cytokeratin peptides identified were mostly unique to one protein (Figure S1). The sequences of the five most abundant cytokeratins identified in the amyloid deposits of the localized cutaneous lichen or macular amyloidosis specimen were aligned [31]. Peptides identified that are not shared are highlighted in red and those identified peptides that are shared are highlighted in blue.

Results of IHC using EPR4287 against galectin-7 on tissue sections from all seven patients were concordant with the results from the mass spectrometric analysis (Figure 3). Galectin-7 was not detected in the amyloid deposits by IHC, but it was detected in the overlying dermis (Figure 3E & F). IHC with 34ßE12 for keratins 1, 5, 10, and 14 highlighted keratins in the amyloid deposits for patients with localized cutaneous lichen amyloidosis and in the overlying dermis regions (Figure 3C & D).

Figure 3.

Amyloid deposits are positive by immunohistochemistry for cytokeratins, but negative for galectin-7. Images of an H&E (A), anti-keratin IHC (C), and anti-galectin-7 IHC (E) stained sections are shown for patient 3. The same images are shown for patient 1 (B, D, F).

Discussion

Analysis using LMD and LCMS is the current gold standard technique for the identification of causative protein in amyloid deposits and this method has been implement in many major institutions [19,23,25,32]. The utilization of microdissection to isolate the amyloid deposits from the larger tissue biopsies allows for a more specific and accurate analysis of the protein composition of the discrete regions of interest. With this approach it is possible to separate the amyloid deposits from the surrounding tissue and analyse the distinct regions independently by mass spectrometry (Figure 1).

When Miura and colleagues detected galectin-7, keratins, and actin in amyloid deposits from a patient with Bowen’s disease they performed protein extraction on amyloid-laden lesional and amyloid-free control skin tissue [12]. However, macro dissection of amyloid deposits can commonly lead to contamination of amyloid deposits with surrounding tissues and therefore the proteins from these tissues. Miura and colleagues excised a band with the molecular weight of 14 kDa from a 2-D gel, digested, and analysed the protein digest by MALDI-TOF mass spectrometer [12].

In our study galectin-7 was detected in all Congo red negative overlying epidermis regions, but in none of the dermal amyloid deposits. LCMS results were filtered to include single peptide or peptide spectral match identifications. If a single peptide from galectin-7 was detected in a sample, it would have been included in the results. Sequence coverage of galectin-7 in the Congo red negative epidermis from our seven-patient cohort was identical to that galectin-7 sequence coverage from Miura and colleagues MALDI-TOF MS analysis (Figure 2) [12]. This implies that if there was galectin-7 present in the amyloid deposits microdissected from our patient cohort it would have been detected in the analysis.

The same total tissue area (50,000 μm²) was microdissected from the Congo red positive amyloid regions and overlying Congo red negative tissue for all samples. All samples were prepared and analysed by LCMS following the clinically validated procedures, so the protein content input was as similar as experimentally possible across patients and samples. This is supported by the consistent presence of actin at comparable levels across all amyloid and non-amyloid tissue samples. Therefore, there is no concern about the amount of protein analysed limiting the detection of galectin-7 in the amyloid deposits.

When IHC for keratins and galectin-7 was performed on the five localized cutaneous lichen or macular amyloidosis samples in our study the galectin-7 antibody was only reactive in the Congo red negative dermis, but the keratin antibody was reactive in both the amyloid deposits and overlying epidermis (Figure 3). In the two nodular cutaneous amyloidosis cases the amyloid deposits were negative for keratin and galectin-7. The overlying Congo red negative epidermis in those two cases were positive for keratin and galectin-7.

Miura and colleagues performed IHC against keratin, galectin-7, and actin on tissue sections from a total of 19 patients with localized cutaneous amyloidosis [12]. They found all tissues to be reactive to all three antibodies in the Congo red positive regions. There is a clear discrepancy between the IHC findings of the two studies. Miura and colleagues used a polyclonal galectin-7 antibody, which is more likely to cross-react with other proteins than a monoclonal antibody such as EPR4287 [12].

Miura and colleagues presented an argument for galectin-7 as the likely causative protein identified in their analysis over keratin based on the native structures of the two proteins [12]. Galectin-7 is composed of pairs of β-strand segments in a parallel formation, but keratins have a predominately alpha helical tertiary structure [33,34]. In 2014 a follow up study was conducted in which attempts were made to form in vitro amyloid fibrils using recombinant proteins and peptides of galectin-7, actin, and keratins [35]. Of the three proteins only, synthetic peptides of galectin-7 formed amyloid fibrils under in vitro induction conditions.

However, many well established amyloidogenic proteins have an alpha helical native structure. For example, serum amyloid A protein exists as a series of alpha helices and readily forms amyloid fibrils [36]. Additionally, Yamada and colleagues have induced in vitro amyloid fibril formation of serum amyloid A protein [37]. Although many amyloidogenic proteins exists in a native β-sheet formation it is not a prerequisite.

Some in the field of amyloidosis research believe there is another protein(s) involved in the formation of localized cutaneous lichen or macular amyloidosis. From the examination of five localized cutaneous lichen or macular amyloidosis cases by LMD paired with LCMS, galectin-7 is not part of the amyloid deposits, but keratin 5 and 14 are present in the deposits. There are potentially additional proteins involved in amyloid fibrils formed in localized cutaneous lichen or macular amyloidosis, but at this time the evidence does not support galectin-7 as the causative protein.

The findings from this study display the importance of laser microdissection for the protein profiling of amyloid deposits and identification of causative proteins. A study utilizing LMD with LCMS of a larger cohort of localized cutaneous lichen or macular amyloidosis cases is necessary to determine if there are alternative or additional causative protein candidates.

Abbreviations:

FFPE

formalin-fixed paraffin-embedded

IHC

immunohistochemistry

LCMS

liquid chromatography – mass spectrometry

LMD

laser microdissection

PSM

peptide spectral match